Sequences of the gene encoding the b-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.The family Pasteurellaceae Pohl 1981 currently comprises 57 named bacterial species which have been isolated from man and various animals. The three genera Haemophilus, Actinobacillus and Pasteurella which originally formed the family have recently been joined by new genera, the most prominent being Mannheimia, which contains the species Mannheimia haemolytica (formerly Pasteurella haemolytica) (Angen et al., 1999). Three genera are currently formed by single species, these are Phocoenobacter uteri (Foster et al., 2000), Lonepinella koalarum (Osawa et al., 1995) and Histophilus somni (Angen et al., 2003). The latter genus has been proposed to include the three species incertae sedis 'Haemophilus somnus', 'Haemophilus agni' and 'Histophilus ovis'. Finally, Christensen et al. (2003a) have described the genus Gallibacterium, which currently consists of one species and one genomospecies.Members of the Pasteurellaceae are generally isolated from mucosal membranes and tissue of birds, turtles and mammals, including man. They show a strong host association and have probably co-evolved with their corresponding hosts (Bisgaard, 1993). While most of the species are commensals, there are a few that act as pathogens . These include the human pathogens Haemophilus influenzae, which causes neonatal meningitis and otitis media, and Actinobacillus actinomycetemcomitans, which causes juvenile periodontitis. Important animal diseases are caused, for example, by M. haemolytica (shipping fever of cattle), Pasteurella multocida (atrophic rhinitis in swine and fowl cholera) and Actinobacillus pleuropneumoniae (pleuropneumonia in pigs). In many of the pathogenic species of Pasteurellaceae, specific toxins belonging to the RTX toxin family are found . These toxins are often associated with specifically pathogenic isolates and seem to determine to some extent the host range of the pathoge...

SUMMARY This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

We present an optimized multilocus sequence typing (MLST) scheme with universal primer sets for amplifying and sequencing the seven target genes of Campylobacter jejuni and Campylobacter coli. Typing was expanded by sequence determination of the genes flaA and flaB using optimized primer sets. This approach is compatible with the MLST and flaA schemes used in the PubMLST database and results in an additional typing method using the flaB gene sequence. An identification module based on the 16S rRNA and rpoB genes was included, as well as the genetic determination of macrolide and quinolone resistances based on mutations in the 23S rRNA and gyrA genes. Experimental procedures were simplified by multiplex PCR of the 13 target genes. This comprehensive approach was evaluated with C. jejuni and C. coli isolates collected in Switzerland. MLST of 329 strains resulted in 72 sequence types (STs) among the 186 C. jejuni strains and 39 STs for the 143 C. coli isolates. Fourteen (19%) of the C. jejuni and 20 (51%) of the C. coli STs had not been found previously. In total, 35% of the C. coli strains collected in Switzerland contained mutations conferring antibiotic resistance only to quinolone, 15% contained mutations conferring resistance only to macrolides, and 6% contained mutations conferring resistance to both classes of antibiotics. In C. jejuni, these values were 31% and 0% for quinolone and macrolide resistance, respectively. The rpoB sequence allowed phylogenetic differentiation between C. coli and C. jejuni, which was not possible by 16S rRNA gene analysis. An online Integrated Database Network System (SmartGene, Zug, Switzerland)-based platform for MLST data analysis specific to Campylobacter was implemented. This Web-based platform allowed automated allele and ST designation, as well as epidemiological analysis of data, thus streamlining and facilitating the analysis workflow. Data networking facilitates the exchange of information between collaborating centers. The described approach simplifies and improves the genotyping of Campylobacter, allowing cost-and time-efficient routine monitoring.

The genus Campylobacter comprises 17 species, some of which are important animal and human pathogens. To gain more insight into the genetic relatedness of this genus and to improve the molecular tools available for diagnosis, a universal sequencing approach was established for the gene encoding the beta-subunit of RNA polymerase (rpoB) for the genus Campylobacter. A total of 59 strains, including the type strains of currently recognized species as well as field isolates, were investigated in the study. A primer set specific for Campylobacter species enabled straightforward amplification and sequencing of a 530 bp fragment of the rpoB gene. The 16S rRNA gene sequences of all of the strains were determined in parallel. A good congruence was obtained between 16S rRNA and rpoB gene sequence-based trees within the genus Campylobacter. The branching of the rpoB tree was similar to that of the 16S rRNA gene tree, even though a few discrepancies were observed for certain species. The resolution of the rpoB gene within the genus Campylobacter was generally much higher than that of the 16S rRNA gene sequence, resulting in a clear separation of most species and even some subspecies. The universally applicable amplification and sequencing approach for partial rpoB gene sequence determination provides a powerful tool for DNA sequence-based discrimination of Campylobacter species.The genus Campylobacter was first proposed by M. Sebald and M. Véron in 1963 and included only the type species Campylobacter fetus and Campylobacter bubulus, now known as Campylobacter sputorum (Sebald & Véron, 1963). These taxa had formerly been classified as Vibrio species. In 1973, M. Véron and R. Chatelain included several misclassified Vibrio in the distinct Campylobacter genus based on serological, biochemical and DNA-DNA hybridization investigations (On, 2001). Since then, the taxonomy of the genus has changed dramatically. At present, it comprises 17 species with validly published names and six recognized subspecies (On, 2001;Foster et al., 2004). In addition, strains belonging to C. sputorum are divided into three biovars, sputorum, fecalis and paraureolyticus, on the basis of their ability to produce catalase or urease Vandamme & On, 2001).In general, members of the genus Campylobacter colonize the mucosal surfaces of the intestinal tract, oral cavity or urogenital tract of healthy, as well as diseased, humans and animals, especially birds. Several species may act as pathogens, causing disease in both human and animal hosts. Twelve of the 17 Campylobacter species are associated with human diseases. Campylobacter jejuni and Campylobacter coli are particularly frequent causative agents of human bacterial intestinal disorders worldwide (Skirrow, 1994). There have also been reported cases of diarrhoea in man caused by Campylobacter upsaliensis and Campylobacter lari, but the frequency of these infections is very low (Bourke et al., 1998;Van Doorn et al., 1998). Occasionally, Campylobacter species are implicated as causative agents of pericard...

There are surely scientific, genetic or ecological arguments which show that differences exist between the relapsing fever (RF) spirochaetes and the Lyme borreliosis (LB) group of spirochaetes, both of which belong to the genus Borrelia. In a recent publication, Adeolu and Gupta [1] proposed dividing the genus Borrelia into two genera on the basis of genetic differences revealed by comparative genomics. The new genus name for the LB group of spirochaetes, Borreliella, has subsequently been entered in the GenBank database for some species of the group and in a validation list (List of new names and new combinations previously effectively, but not validly, published) [2]. However, rapidly expanding scientific knowledge and considerable conflicting evidence combined with the adverse consequences of splitting the genus Borrelia make such a drastic step somewhat premature. In our opinion, the basis of this division rests on preliminary evidence and should be rescinded for the following reasons:(1) The proposed split of the genus rests on differences in conserved signature indels (CSI) and conserved signature proteins (CSP) between LB and RF spirochaetes. A major omission in the study published by Adeolu and Gupta [1] is the exclusion of a Borrelia clade containing RF-like species that utilize hard ticks as vectors and reptiles as reservoir hosts [3,4].To identify proteins that are uniquely present in various groups of Borrelia, BLAST searches [5] were performed by Adeolu and Gupta [1] using each protein in the genomes of Borrelia burgdorferi sensu stricto (s.s.) B31 T and Borrelia recurrentis A1 as queries. Out of 1041 and 1390 protein coding genes (i.e. the number of proteins reported in GenBank accession numbers NC_011244 and NC_001318) present in B. recurrentis A1 and B. burgdorferi s.s. B31 T , respectively, 15 CSI (seven for LB, eight for RF) and 25 CSP (21 for LB, four for RF) were found to be unique for the respective groups. However, two of the four CSPs that are apparently unique for the RF group species are not found in all members of this group and therefore do not represent true signature proteins. Hence, just two CSPs and eight CSIs are unique to the RF group.