Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Prediction of whole-genome DNA-DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA-DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA-DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA-DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA-DNA hybridization.

Phylogeny and prediction of genetic similarity of Cronobacter and related taxa by multilocus sequence analysis (MLSA)

Multilocus sequence analysis (MLSA) based on recN, rpoA and thdF genes was done on more than 30 species of the family Enterobacteriaceae with a focus on Cronobacter and the related genus Enterobacter. The sequences provide valuable data for phylogenetic, taxonomic and diagnostic purposes. Phylogenetic analysis showed that the genus Cronobacter forms a homogenous cluster related to recently described species of Enterobacter, but distant to other species of this genus. Combining sequence information on all three genes is highly representative for the species' %GC-content used as taxonomic marker. Sequence similarity of the three genes and even of recN alone can be used to extrapolate genetic similarities between species of Enterobacteriaceae. Finally, the rpoA gene sequence, which is the easiest one to determine, provides a powerful diagnostic tool to identify and differentiate species of this family. The comparative analysis gives important insights into the phylogeny and genetic relatedness of the family Enterobacteriaceae and will serve as a basis for further studies and clarifications on the taxonomy of this large and heterogeneous family.

Multiplex strategy for multilocus sequence typing, fla typing, and genetic determination of antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates collected in Switzerland

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.

Multilocus sequence typing (MLST) of Mycoplasma hyopneumoniae: a diverse pathogen with limited clonality

A multilocus sequence typing (MLST) scheme was established and evaluated for Mycoplasma hyopneumoniae, the etiologic agent of enzootic pneumonia in swine with the aim of defining strains. Putative target genes were selected by genome sequence comparisons. Out of 12 housekeeping genes chosen and experimentally validated, the 7 genes efp, metG, pgiB, recA, adk, rpoB, and tpiA were finally used to establish the MLST scheme. Their usefulness was assessed individually and in combination using a set of well-defined field samples and strains of M. hyopneumoniae. A reduction to the three targets showing highest variation (adk, rpoB, and tpiA) was possible resulting in the same number of sequence types as using the seven targets. The established MLST approach was compared with the recently described typing method using the serine-rich repeat motif-encoding region of the p146 gene. There was coherence between the two methods, but MLST resulted in a slightly higher resolution. Farms recognized to be affected by enzootic pneumonia were always associated with a single M. hyopneumoniae clone, which in most cases differed from farm to farm. However, farms in close geographic or operational contact showed identical clones as defined by MLST typing. Population analysis showed that recombination in M. hyopneumoniae occurs and that strains are very diverse with only limited clonality observed. Elaborate classical MLST schemes using multiple targets for M. hyopneumoniae might therefore be of limited value. In contrast, MLST typing of M. hyopneumoniae using the three genes adk, rpoB, and tpiA seems to be sufficient for epidemiological investigations by direct amplification of target genes from lysate of clinical material without prior cultivation.

Comparative human-horse sequence analysis of the CYP3A subfamily gene cluster

Cytochrome P450 enzymes (CYP450s) represent a superfamily of haem-thiolate proteins. CYP450s are most abundant in the liver, a major site of drug metabolism, and play key roles in the metabolism of a variety of substrates, including drugs and environmental contaminants. Interaction of two or more different drugs with the same enzyme can account for adverse effects and failure of therapy. Human CYP3A4 metabolizes about 50% of all known drugs, but little is known about the orthologous CYP450s in horses. We report here the genomic organization of the equine CYP3A gene cluster as well as a comparative analysis with the human CYP3A gene cluster. The equine CYP450 genes of the 3A family are located on ECA 13 between 6.97-7.53 Mb, in a region syntenic to HSA 7 99.05-99.35 Mb. Seven potential, closely linked equine CYP3A genes were found, in contrast to only four genes in the human genome. RNA was isolated from an equine liver sample, and the approximately 1.5-kb coding sequence of six CYP3A genes could be amplified by RT-PCR. Sequencing of the RT-PCR products revealed numerous hitherto unknown single nucleotide polymorphisms (SNPs) in these six CYP3A genes, and one 6-bp deletion compared to the reference sequence (EquCab2.0). The presence of the variants was confirmed in a sample of genomic DNA from the same horse. In conclusion, orthologous genes for the CYP3A family exist in horses, but their number differs from those of the human CYP3A gene family. CYP450 genes of the same family show high homology within and between mammalian species, but can be highly polymorphic.

Sequence analysis of the porcine IFNAR1 and IFNGR2 genes

A porcine BAC clone harboring the tightly linked IFNAR1 and IFNGR2 genes was identified by comparative analysis of the publicly available porcine BAC end sequences. The complete 168,835 bp insert sequence of this clone was determined. Sequence comparisons of the genomic sequence with EST sequences from public databases were performed and allowed a detailed annotation of the IFNAR1 and IFNGR2 genes. The analyzed genes showed a conserved genomic organization with their known mammalian orthologs, however the sequence conservation of these genes across species was relatively low. In addition to the IFNAR1 and IFNGR2 genes, which were completely sequenced, the analyzed BAC clone also contained parts of an orphan gene encoding a putative transmembrane protein (TMEM50B). In contrast to the IFNAR1 and IFNGR2 genes the sequence conservation of the TMEM50B gene across different mammalian species was extremely high.

Sequence analysis of a 212 kb defensin gene cluster on ECA 27q17

Defensins are a family of evolutionary ancient antimicrobial peptides consisting of three sub-families: alpha-, beta- and theta-defensins. This investigation was focused on the genomic characterization of equine beta-defensins and the investigation of the potential clustering of beta-defensin genes in the equine genome. Six genomic BAC clones were isolated from the CHORI-241 library and one of these was mapped by FISH to ECA 27q17. This location was confirmed by RH-mapping. The contiguous 212 kb sequence of this clone was determined. Sequence analysis revealed the identification of ten pseudogenes and nine genes, six of which were highly homologous to human beta-defensin DEFB4. Clustering of the beta-defensin genes was confirmed and the order of the genes on the analyzed BAC was related to the corresponding defensin cluster on HSA 8. The knowledge about the sequence and the genomic structure of the equine beta-defensin genes will improve the classification of different paralogous defensin genes and is a prerequisite for subsequent functional studies. Additionally, the first alpha-defensin-like sequence outside the groups of primates, lagomorphs and rodents (glires) was identified.

Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes

We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.

Multilocus sequence typing for characterization of Staphylococcus pseudintermedius

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.

Streaming Support for Data Intensive Cloud-Based Sequence Analysis

Cloud computing provides a promising solution to the genomics data deluge problem resulting from the advent of next-generation sequencing (NGS) technology. Based on the concepts of “resources-on-demand” and “pay-as-you-go”, scientists with no or limited infrastructure can have access to scalable and cost-effective computational resources. However, the large size of NGS data causes a significant data transfer latency from the client’s site to the cloud, which presents a bottleneck for using cloud computing services. In this paper, we provide a streaming-based scheme to overcome this problem, where the NGS data is processed while being transferred to the cloud. Our scheme targets the wide class of NGS data analysis tasks, where the NGS sequences can be processed independently from one another. We also provide the elastream package that supports the use of this scheme with individual analysis programs or with workflow systems. Experiments presented in this paper show that our solution mitigates the effect of data transfer latency and saves both time and cost of computation.

Assessing the Potential of Multiple Imputation in Sequence Analysis

Sequence analysis and optimal matching are useful heuristic tools for the descriptive analysis of heterogeneous individual pathways such as educational careers, job sequences or patterns of family formation. However, to date it remains unclear how to handle the inevitable problems caused by missing values with regard to such analysis. Multiple Imputation (MI) offers a possible solution for this problem but it has not been tested in the context of sequence analysis. Against this background, we contribute to the literature by assessing the potential of MI in the context of sequence analyses using an empirical example. Methodologically, we draw upon the work of Brendan Halpin and extend it to additional types of missing value patterns. Our empirical case is a sequence analysis of panel data with substantial attrition that examines the typical patterns and the persistence of sex segregation in school-to-work transitions in Switzerland. The preliminary results indicate that MI is a valuable methodology for handling missing values due to panel mortality in the context of sequence analysis. MI is especially useful in facilitating a sound interpretation of the resulting sequence types.

Proposal of Bisgaardia hudsonensis gen. nov., sp. nov. and an additional genomospecies, isolated from seals, as new members of the family Pasteurellaceae

Phenotypic and phylogenetic studies were performed on eight Gram-negative-staining, rod-shaped bacteria isolated from seals. Biochemical and physiological studies showed identical profiles for all of the isolates and indicated that they were related to the family Pasteurellaceae. 16S rRNA gene sequencing demonstrated that the organism represented a distinct cluster with two sublines within the family Pasteurellaceae with <96% sequence similarity to any recognized species. Multilocus sequence analysis (MLSA) including rpoB, infB and recN genes further confirmed these findings with the eight isolates forming a genus-like cluster with two branches. Genome relatedness as deduced from recN gene sequences suggested that the isolates represented a new genus with two species. On the basis of the results of the phylogenetic analysis and phenotypic criteria, it is proposed that these bacteria from seals are classified as Bisgaardia hudsonensis gen. nov., sp. nov. (the type species) and Bisgaardia genomospecies 1. The G+C content of the DNA was 39.5 mol%. The type strain of Bisgaardia hudsonensis gen. nov., sp. nov. is M327/99/2(T) (=CCUG 43067(T)=NCTC 13475(T)=98-D-690B(T)) and the reference strain of Bisgaardia genomospecies 1 is M1765/96/5 (=CCUG 59551=NCTC 13474).

Basfia succiniciproducens gen. nov., sp. nov., a new member of the family Pasteurellaceae isolated from bovine rumen

Gram-negative, coccoid, non-motile bacteria that are catalase-, urease- and indole-negative, facultatively anaerobic and oxidase-positive were isolated from the bovine rumen using an improved selective medium for members of the Pasteurellaceae. All strains produced significant amounts of succinic acid under anaerobic conditions with glucose as substrate. Phenotypic characterization and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, infB and recN genes were performed on seven independent isolates. All four genes showed high sequence similarity to their counterparts in the genome sequence of the patent strain MBEL55E, but less than 95 % 16S rRNA gene sequence similarity to any other species of the Pasteurellaceae. Genetically these strains form a very homogeneous group in individual as well as combined phylogenetic trees, clearly separated from other genera of the family from which they can also be separated based on phenotypic markers. Genome relatedness as deduced from the recN gene showed high interspecies similarities, but again low similarity to any of the established genera of the family. No toxicity towards bovine, human or fish cells was observed and no RTX toxin genes were detected in members of the new taxon. Based on phylogenetic clustering in the MLSA analysis, the low genetic similarity to other genera and the phenotypic distinction, we suggest to classify these bovine rumen isolates as Basfia succiniciproducens gen. nov., sp. nov. The type strain is JF4016(T) (=DSM 22022(T) =CCUG 57335(T)).