Global dissemination of a multidrug resistant Escherichia coli clone

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000–2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL–resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.

Chaperone-usher fimbriae of Escherichia coli

Chaperone-usher (CU) fimbriae are adhesive surface organelles common to many Gram-negative bacteria. Escherichia coli genomes contain a large variety of characterised and putative CU fimbrial operons, however, the classification and annotation of individual loci remains problematic. Here we describe a classification model based on usher phylogeny and genomic locus position to categorise the CU fimbrial types of E. coli. Using the BLASTp algorithm, an iterative usher protein search was performed to identify CU fimbrial operons from 35 E. coli (and one Escherichia fergusonnii) genomes representing different pathogenic and phylogenic lineages, as well as 132 Escherichia spp. plasmids. A total of 458 CU fimbrial operons were identified, which represent 38 distinct fimbrial types based on genomic locus position and usher phylogeny. The majority of fimbrial operon types occupied a specific locus position on the E. coli chromosome; exceptions were associated with mobile genetic elements. A group of core-associated E. coli CU fimbriae were defined and include the Type 1, Yad, Yeh, Yfc, Mat, F9 and Ybg fimbriae. These genes were present as intact or disrupted operons at the same genetic locus in almost all genomes examined. Evaluation of the distribution and prevalence of CU fimbrial types among different pathogenic and phylogenic groups provides an overview of group specific fimbrial profiles and insight into the ancestry and evolution of CU fimbriae in E. coli.

Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transferase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes, GSTT1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GSTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals, e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestive tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

The long and short of it: branch lengths and the problem of placing the New Zealand short-tailed bat.

The taxonomic position of the endemic New Zealand bat genus Mystacina has vexed systematists ever since its erection in 1843. Over the years the genus has been linked with many microchiropteran families and superfamilies. Most recent classifications place it in the Vespertilionoidea, although some immunological evidence links it with the Noctilionoidea (=Phyllostomoidea). We have sequenced 402 bp of the mitochondrial cytochrome b gene for M. tuberculata (Gray in Dieffenbach, 1843), and using both our own and published DNA sequences for taxa in both superfamilies, we applied different tree reconstruction methods to find the appropriate phylogeny and different methods of estimating confidence in the parts of the tree. All methods strongly support the classification of Mystacina in the Noctilionoidea. Spectral analysis suggests that parsimony analysis may be misleading for Mystacina's precise placement within the Noctilionoidea because of its long terminal branch. Analyses not susceptible to long-branch attraction suggest that the Mystacinidae is a sister family to the Phyllostomidae. Dating the divergence times between the different taxa suggests that the extant chiropteran families radiated around and shortly after the Cretaceous–Tertiary boundary. We discuss the biogeographical implications of classifying Mystacina within the Noctilionoidea and contrast our result with those classifications placing Mystacina in the Vespertilionoidea, concluding that evidence for the latter is weak.

The Long and Short of It: Branch Lengths and the Problem of Placing the New Zealand Short-Tailed Bat, Mystacina

The taxonomic position of the endemic New Zealand bat genus Mystacina has vexed systematists ever since its erection in 1843. Over the years the genus has been linked with many microchiropteran families and superfamilies. Most recent classifications place it in the Vespertilionoidea, although some immunological evidence links it with the Noctilionoidea (=Phyllostomoidea). We have sequenced 402 bp of the mitochondrial cytochrome b gene for M. tuberculata (Gray in Dieffenbach, 1843), and using both our own and published DNA sequences for taxa in both superfamilies, we applied different tree reconstruction methods to find the appropriate phylogeny and different methods of estimating confidence in the parts of the tree. All methods strongly support the classification of Mystacina in the Noctilionoidea. Spectral analysis suggests that parsimony analysis may be misleading for Mystacina's precise placement within the Noctilionoidea because of its long terminal branch. Analyses not susceptible to long-branch attraction suggest that the Mystacinidae is a sister family to the Phyllostomidae. Dating the divergence times between the different taxa suggests that the extant chiropteran families radiated around and shortly after the Cretaceous-Tertiary boundary. We discuss the biogeographical implications of classifying Mystacina within the Noctilionoidea and contrast our result with those classifications placing Mystacina in the Vespertilionoidea, concluding that evidence for the latter is weak.

The evolutionary history of termites as inferred from 66 mitochondrial genomes

Termites have colonized many habitats and are among the most abundant animals in tropical ecosystems, which they modify considerably through their actions. The timing of their rise in abundance and of the dispersal events that gave rise to modern termite lineages is not well understood. To shed light on termite origins and diversification, we sequenced the mitochondrial genome of 48 termite species and combined them with 18 previously sequenced termite mitochondrial genomes for phylogenetic and molecular clock analyses using multiple fossil calibrations. The 66 genomes represent most major clades of termites. Unlike previous phylogenetic studies based on fewer molecular data, our phylogenetic tree is fully resolved for the lower termites. The phylogenetic positions of Macrotermitinae and Apicotermitinae are also resolved as the basal groups in the higher termites, but in the crown termitid groups, including Termitinae + Syntermitinae + Nasutitermitinae + Cubitermitinae, the position of some nodes remains uncertain. Our molecular clock tree indicates that the lineages leading to termites and Cryptocercus roaches diverged 170 Ma (153-196 Ma 95% confidence interval [CI]), that modern Termitidae arose 54 Ma (46-66 Ma 95% CI), and that the crown termitid group arose 40 Ma (35-49 Ma 95% CI). This indicates that the distribution of basal termite clades was influenced by the final stages of the breakup of Pangaea. Our inference of ancestral geographic ranges shows that the Termitidae, which includes more than 75% of extant termite species, most likely originated in Africa or Asia, and acquired their pantropical distribution after a series of dispersal and subsequent diversification events.

The origins and radiation of Australian Coptotermes termites: From rainforest to desert dwellers

The termite genus Coptotermes (Rhinotermitidae) is found in Asia, Africa, Central/South America and Australia, with greatest diversity in Asia. Some Coptotermes species are amongst the world’s most damaging invasive termites, but the genus is also significant for containing the most sophisticated mound-building termites outside the family Termitidae. These mound-building Coptotermes occur only in Australia. Despite its economic and evolutionary significance, the biogeographic history of the genus has not been well investigated, nor has the evolution of the Australian mound-building species. We present here the first phylogeny of the Australian Coptotermes to include representatives from all described species. We combined our new data with previously generated data to estimate the first phylogeny to include representatives from all continents where the genus is found. We also present the first estimation of divergence dates during the evolution of the genus. We found the Australian Coptotermes to be monophyletic and most closely related to the Asian Coptotermes, with considerable genetic diversity in some Australian taxa possibly representing undescribed species. The Australian mound-building species did not form a monophyletic clade. Our ancestral state reconstruction analysis indicated that the ancestral Australian Coptotermes was likely to have been a tree nester, and that mound-building behaviour has arisen multiple times. The Australian Coptotermes were found to have diversified ∼13 million years ago, which plausibly matches with the narrowing of the Arafura Sea allowing Asian taxa to cross into Australia. The first diverging Coptotermes group was found to be African, casting doubt on the previously raised hypothesis that the genus has an Asian origin.

Four mammal fossil calibrations: Balancing competing palaeontological and molecular considerations

With the introduction of relaxed-clock molecular dating methods, the role of fossil calibration has expanded from providing a timescale, to also informing the models for molecular rate variation across the phylogeny. Here I suggest fossil calibration bounds for four mammal clades, Monotremata (platypus and echidnas), Macropodoidea (kangaroos and potoroos), Caviomorpha-Phiomorpha (South American and African hystricognath rodents), and Chiroptera (bats). In each case I consider sources of uncertainty in the fossil record and provide a molecular dating analysis to examine how the suggested calibration priors are further informed by other mammal fossil calibrations and molecular data.

Comparative analysis of genome maintenance genes in naked mole rat, mouse, and human

Genome maintenance (GM) is an essential defense system against aging and cancer, as both are characterized by increased genome instability. Here, we compared the copy number variation and mutation rate of 518 GM-associated genes in the naked mole rat (NMR), mouse, and human genomes. GM genes appeared to be strongly conserved, with copy number variation in only four genes. Interestingly, we found NMR to have a higher copy number of CEBPG, a regulator of DNA repair, and TINF2, a protector of telomere integrity. NMR, as well as human, was also found to have a lower rate of germline nucleotide substitution than the mouse. Together, the data suggest that the long-lived NMR, as well as human, has more robust GM than mouse and identifies new targets for the analysis of the exceptional longevity of the NMR.

Comparative analysis and distribution of Omega-3 lcPUFA Biosynthesis genes in marine molluscs

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs.

Resampling-based approaches to study variation in morphological modularity

Modularity has been suggested to be connected to evolvability because a higher degree of independence among parts allows them to evolve as separate units. Recently, the Escoufier RV coefficient has been proposed as a measure of the degree of integration between modules in multivariate morphometric datasets. However, it has been shown, using randomly simulated datasets, that the value of the RV coefficient depends on sample size. Also, so far there is no statistical test for the difference in the RV coefficient between a priori defined groups of observations. Here, we (1), using a rarefaction analysis, show that the value of the RV coefficient depends on sample size also in real geometric morphometric datasets; (2) propose a permutation procedure to test for the difference in the RV coefficient between a priori defined groups of observations; (3) show, through simulations, that such a permutation procedure has an appropriate Type I error; (4) suggest that a rarefaction procedure could be used to obtain sample-size-corrected values of the RV coefficient; and (5) propose a nearest-neighbor procedure that could be used when studying the variation of modularity in geographic space. The approaches outlined here, readily extendable to non-morphometric datasets, allow study of the variation in the degree of integration between a priori defined modules. A Java application – that will allow performance of the proposed test using a software with graphical user interface – has also been developed and is available at the Morphometrics at Stony Brook Web page (http://life.bio.sunysb.edu/morph/).

Bat species comparisons based on external morphology: A test of traditional versus geometric morphometric approaches

External morphology is commonly used to identify bats as well as to investigate flight and foraging behavior, typically relying on simple length and area measures or ratios. However, geometric morphometrics is increasingly used in the biological sciences to analyse variation in shape and discriminate among species and populations. Here we compare the ability of traditional versus geometric morphometric methods in discriminating between closely related bat species – in this case European horseshoe bats (Rhinolophidae, Chiroptera) – based on morphology of the wing, body and tail. In addition to comparing morphometric methods, we used geometric morphometrics to detect interspecies differences as shape changes. Geometric morphometrics yielded improved species discrimination relative to traditional methods. The predicted shape for the variation along the between group principal components revealed that the largest differences between species lay in the extent to which the wing reaches in the direction of the head. This strong trend in interspecific shape variation is associated with size, which we interpret as an evolutionary allometry pattern.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.

Interleukin 17A is an immune marker for chlamydial disease severity and pathogenesis in the koala (Phascolarctos cinereus)

The koala (Phascolarctos cinereus) is an iconic Australian marsupial species that is facing many threats to its survival. Chlamydia pecorum infections are a significant contributor to this ongoing decline. A major limiting factor in our ability to manage and control chlamydial disease in koalas is a limited understanding of the koala’s cell-mediated immune response to infections by this bacterial pathogen. To identify immunological markers associated with chlamydial infection and disease in koalas, we used koala-specific Quantitative Real Time PCR (qrtPCR) assays to profile the cytokine responses of Peripheral Blood Mononuclear Cells (PBMCs) collected from 41 koalas with different stages of chlamydial disease. Target cytokines included the principal Th1 (Interferon gamma; IFNγ), Th2 (Interleukin 10; IL10), and pro-inflammatory cytokines (Tumor Necrosis Factor alpha; TNFα). A novel koala-specific IL17A qrtPCR assay was also developed as part of this study to quantitate the gene expression of this Th17 cytokine in koalas. A statistically significant higher IL17A gene expression was observed in animals with current chlamydial disease compared to animals with asymptomatic chlamydial infection. A modest up-regulation of pro-inflammatory cytokines, such as TNFα and IFNγ, was also observed in these animals with signs of current chlamydial disease. IL10 gene expression was not evident in the majority of animals from both groups. Future longitudinal studies are now required to confirm the role played by cytokines in pathology and/or protection against C. pecorum infection in the koala.

Structure and vascular tissue expression of duplicated TERMINAL EAR1-like paralogues in poplar

TERMINAL EAR1-like (TEL) genes encode putative RNA-binding proteins only found in land plants. Previous studies suggested that they may regulate tissue and organ initiation in Poaceae. Two TEL genes were identified in both Populus trichocarpa and the hybrid aspen Populus tremula × P. alba, named, respectively, PoptrTEL1-2 and PtaTEL1-2. The analysis of the organisation around the PoptrTEL genes in the P. trichocarpa genome and the estimation of the synonymous substitution rate for PtaTEL1-2 genes indicate that the paralogous link between these two Populus TEL genes probably results from the Salicoid large-scale gene-duplication event. Phylogenetic analyses confirmed their orthology link with the other TEL genes. The expression pattern of both PtaTEL genes appeared to be restricted to the mother cells of the plant body: leaf founder cells, leaf primordia, axillary buds and root differentiating tissues, as well as to mother cells of vascular tissues. Most interestingly, PtaTEL1-2 transcripts were found in differentiating cells of secondary xylem and phloem, but probably not in the cambium itself. Taken together, these results indicate specific expression of the TEL genes in differentiating cells controlling tissue and organ development in Populus (and other Angiosperm species).