Molecular characterisation of environmental enterococci derived from water samples and assessment of associated public health hazards

Enterococci are versatile Gram-positive bacteria that can survive under extreme conditions. Most enterococci are non-virulent and found in the gastrointestinal tract of humans and animals. Other strains are opportunistic pathogens that contribute to a large number of nosocomial infections globally. Epidemiological studies demonstrated a direct relationship between the density of enterococci in surface waters and the risk of swimmer-associated gastroenteritis. The distribution of infectious enterococcal strains from the hospital environment or other sources to environmental water bodies through sewage discharge or other means, could increase the prevalence of these strains in the human population. Environmental water quality studies may benefit from focusing on a subset of Enterococcus spp. that are consistently associated with sources of faecal pollution such as domestic sewage, rather than testing for the entire genus. E. faecalis and E. faecium are potentially good focal species for such studies, as they have been consistently identified as the dominant Enterococcus spp. in human faeces and sewage. On the other hand enterococcal infections are predominantly caused by E. faecalis and E. faecium. The characterisation of E. faecalis and E. faecium is important in studying their population structures, particularly in environmental samples. In developing and implementing rapid, robust molecular genotyping techniques, it is possible to more accurately establish the relationship between human and environmental enterococci. Of particular importance, is to determine the distribution of high risk enterococcal clonal complexes, such as E. faecium clonal complex 17 and E. faecalis clonal complexes 2 and 9 in recreational waters. These clonal complexes are recognized as particularly pathogenic enterococcal genotypes that cause severe disease in humans globally. The Pimpama-Coomera watershed is located in South East Queensland, Australia and was investigated in this study mainly because it is used intensively for agriculture and recreational purposes and has a strong anthropogenic impact. The primary aim of this study was to develop novel, universally applicable, robust, rapid and cost effective genotyping methods which are likely to yield more definitive results for the routine monitoring of E. faecalis and E. faecium, particularly in environmental water sources. To fullfill this aim, new genotyping methods were developed based on the interrogation of highly informative single nucleotide polymorphisms (SNPs) located in housekeeping genes of both E. faecalis and E. faecium. SNP genotyping was successfully applied in field investigations of the Coomera watershed, South-East Queensland, Australia. E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles respectively. This study showed the high longitudinal diversity of E. faecalis and E. faecium over a period of two years, and both human-related and human-specific SNP profiles were identified. Furthermore, 4.25% of E. faecium strains isolated from water was found to correspond to the important clonal complex-17 (CC17). Strains that belong to CC17 cause the majority of hospital outbreaks and clinical infections globally. Of the six sampling sites of the Coomera River, Paradise Point had the highest number of human-related and human-specific E. faecalis and E. faecium SNP profiles. The secondary aim of this study was to determine the antibiotic-resistance profiles and virulence traits associated with environmental E. faecalis and E. faecium isolates compared to human pathogenic E. faecalis and E. faecium isolates. This was performed to predict the potential health risks associated with coming into contact with these strains in the Coomera watershed. In general, clinical isolates were found to be more resistant to all the antibiotics tested compared to water isolates and they harbored more virulence traits. Multi-drug resistance was more prevalent in clinical isolates (71.18% of E. faecalis and 70.3 % of E. faecium) compared to water isolates (only 5.66 % E. faecium). However, tetracycline, gentamicin, ciprofloxacin and ampicillin resistance was observed in water isolates. The virulence gene esp was the most prevalent virulence determinant observed in clinical isolates (67.79% of E. faecalis and 70.37 % of E. faecium), and this gene has been described as a human-specific marker used for microbial source tracking (MST). The presence of esp in water isolates (16.36% of E. faecalis and 19.14% of E. faecium) could be indicative of human faecal contamination in these waterways. Finally, in order to compare overall gene expression between environmental and clinical strains of E. faecalis, a comparative gene hybridization study was performed. The results of this investigation clearly demonstrated the up-regulation of genes associated with pathogenicity in E. faecalis isolated from water. The expression study was performed at physiological temperatures relative to ambient temperatures. The up-regulation of virulence genes demonstrates that environmental strains of E. faecalis can pose an increased health risk which can lead to serious disease, particularly if these strains belong to the virulent CC17 group. The genotyping techniques developed in this study not only provide a rapid, robust and highly discriminatory tool to characterize E. faecalis and E. faecium, but also enables the efficient identification of virulent enterococci that are distributed in environmental water sources.

Orthopedic bone plates : evolution in structure implementation technique and biomaterial

With many important developments over the last century, nowadays orthopedic bone plate now excels over other types of internal fixators in bone fracture fixation. The developments involve the design, material and implementation techniques of the plates. This paper aims to review the evolution in implementation technique and biomaterial of the orthopedic bone plates. Plates were initially used to fix the underlying bones firmly. Accordingly, Compression plate (CP), Dynamic compression plate (DCP), Limited contact dynamic compression plate (LC-DCP) and Point contact fixator (PC-Fix) were developed. Later, the implementation approach was changed to locking, and the Less Invasive Stabilization System (LISS) plate was introduced as a result. Finally, a combination of both of these approaches has been used by introducing the Locking Compression Plate (LCP). Currently, precontoured LCPs are mainly used for bone fracture fixation. In parallel with structure and implementation techniques, numerous advances have occurred in biomaterials of the plates. Titanium and stainless steel alloys are now the most common biomaterials in production of orthopedic bone plates. However, regarding the biocompatibility, bioactivity and biodegradability characteristics of Mg alloys, Ta alloys, SMAs, carbon fiber composites and bioceramics, these materials are considered as potentially suitable for plates. However, due to poor mechanical properties, they have very limited applications. Therefore, further studies are required in future to solve these problems and make them feasible for heavy-duty bone plates.

Insights into historical drainage evolution based on the phylogeography of the chevron snakehead fish (Channa striata) in the Mekong Basin

1. The phylogeography of freshwater taxa is often integrally linked with landscape changes such as drainage re-alignments that may present the only avenue for historical dispersal for these taxa. Classical models of gene flow do not account for landscape changes and so are of little use in predicting phylogeography in geologically young freshwater landscapes. When the history of drainage formation is unknown, phylogeographical predictions can be based on current freshwater landscape structure, proposed historical drainage geomorphology, or from phylogeographical patterns of co-distributed taxa. 2. This study describes the population structure of a sedentary freshwater fish, the chevron snakehead (Channa striata), across two river drainages on the Indochinese Peninsula. The phylogeographical pattern recovered for C. striata was tested against seven hypotheses based on contemporary landscape structure, proposed history and phylogeographical patterns of codistributed taxa. 3. Consistent with the species ecology, analysis of mitochondrial and microsatellite loci revealed very high differentiation among all sampled sites. A strong signature of historical population subdivision was also revealed within the contemporary Mekong River Basin (MRB). Of the seven phylogeographical hypotheses tested, patterns of co-distributed taxa proved to be the most adequate for describing the phylogeography of C. striata. 4. Results shed new light on SE Asian drainage evolution, indicating that the Middle MRB probably evolved via amalgamation of at least three historically independent drainage sections and in particular that the Mekong River section centred around the northern Khorat Plateau in NE Thailand was probably isolated from the greater Mekong for an extensive period of evolutionary time. In contrast, C. striata populations in the Lower MRB do not show a phylogeographical signature of evolution in historically isolated drainage lines, suggesting drainage amalgamation has been less important for river landscape formation in this region.

Emergent innovation through the co-evolution of informal and formal media economies

Well-established distinctions between amateur and professional are blurring as the impact of social media, changes in cultural consumption, and crises in copyright industries’ business models are felt across society and economy. I call this the increasingly rapid co-evolution of the formal market and informal household sectors and analyse it through the concept of ‘social network markets’ – individual choices are made on the basis of other’s choices and such networked preferencing is enhanced by the growing ubiquity of social media platforms. This may allow us better to understand sources of disruption and innovation in audiovisual production and distribution in wealthy Western markets which are as significant as those posed by informal practices outside the West. I examine what is happening around the monetization and professionalization of online video (YouTube, for example) and the socialization of professional production strategies (transmedia, for example) as innovation from the margins.

The evolution of QUT’s Student Success Program : 20,000 students later

“The Student Success Program (SSP) is a monitoring and early intervention program in operation at QUT designed to identify and support those students deemed to be at risk of disengaging for their learning and their institution” (Nelson, Quinn, Marrington & Clarke, 2011, p. 83). This report reflects on the development of the program since its inception in 2007. In acknowledging similar initiatives within the sector that monitor student learning engagement, the Nuts & Bolts session allows for identification and discussion of the critical success factors for these intervention and support programs.

An expressed sequence tag (EST) library for Drosophila serrata, a model system for sexual selection and climatic adaptation studies

The native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST) collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup.

Polyandry and paternity skew in natural and experimental populations of Drosophila serrata

Many species engage in polyandry, resulting in the potential for sexual selection to continue post-copulation through sperm competition and/or cryptic female choice. The relative importance of pre- vs. post-copulatory processes remains unknown for most species despite this information being fundamental for understanding the evolutionary consequences of sexual selection. The Australian fruit fly Drosophila serrata has become a prominent model system for studying precopulatory sexual selection, such as mating preferences and their influence on the evolution of sexually selected traits. Here, we investigated polyandry and the potential for post-copulatory sexual selection in this species using indirect paternity analysis. We genotyped 21 wild-caught and 19 laboratory-reared mothers and their offspring (a total of 787 flies) at six microsatellite loci and found extensive polyandry, with all broods surveyed having at least two sires. Female remating rates were higher than in other Drosophila surveyed to date and no significant differences were found between laboratory and field populations. Additionally, we found evidence for biased sperm usage in several broods of D. serrata. Paternity skew occurred more frequently in broods from the field population than the laboratory one, suggesting differences between the two environments in the level of post-copulatory sexual selection. Our data suggest that D. serrata represents a promising system for studying the interaction between pre- and post-copulatory sexual selection in driving the evolution of sexually selected phenotypes.

Gene duplication is an evolutionary mechanism for expanding spectral diversity in the long-wavelength photopigments of butterflies

Butterfly long-wavelength (L) photopigments are interesting for comparative studies of adaptive evolution because of the tremendous phenotypic variation that exists in their wavelength of peak absorbance (lambda(max) value). Here we present a comprehensive survey of L photopigment variation by measuring lambda(max) in 12 nymphalid and 1 riodinid species using epi-microspectrophotometry. Together with previous data, we find that L photopigment lambda(max) varies from 510-565 nm in 22 nymphalids, with an even broader 505- to 600-nm range in riodinids. We then surveyed the L opsin genes for which lambda(max) values are available as well as from related taxa and found 2 instances of L opsin gene duplication within nymphalids, in Hermeuptychia hermes and Amathusia phidippus, and 1 instance within riodinids, in the metalmark butterfly Apodemia mormo. Using maximum parsimony and maximum likelihood ancestral state reconstructions to map the evolution of spectral shifts within the L photopigments of nymphalids, we estimate the ancestral pigment had a lambda(max) = 540 nm +/- 10 nm standard error and that blueshifts in wavelength have occurred at least 4 times within the family. We used ancestral state reconstructions to investigate the importance of several amino acid substitutions (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) previously shown to have evolved under positive selection that are correlated with blue spectral shifts. These reconstructions suggest that the Ala64Ser substitution has indeed occurred along the newly identified blueshifted L photopigment lineages. Substitutions at the other 3 sites may also be involved in the functional diversification of L photopigments. Our data strongly suggest that there are limits to the evolution of L photopigment spectral shifts among species with only one L opsin gene and that opsin gene duplication broadens the potential range of lambda(max) values.

Adaptive evolution of color vision as seen through the eyes of butterflies

Butterflies and primates are interesting for comparative color vision studies, because both have evolved middle- (M) and long-wavelength- (L) sensitive photopigments with overlapping absorbance spectrum maxima (lambda(max) values). Although positive selection is important for the maintenance of spectral variation within the primate pigments, it remains an open question whether it contributes similarly to the diversification of butterfly pigments. To examine this issue, we performed epimicrospectrophotometry on the eyes of five Limenitis butterfly species and found a 31-nm range of variation in the lambda(max) values of the L-sensitive photopigments (514-545 nm). We cloned partial Limenitis L opsin gene sequences and found a significant excess of replacement substitutions relative to polymorphisms among species. Mapping of these L photopigment lambda(max) values onto a phylogeny revealed two instances within Lepidoptera of convergently evolved L photopigment lineages whose lambda(max) values were blue-shifted. A codon-based maximum-likelihood analysis indicated that, associated with the two blue spectral shifts, four amino acid sites (Ile17Met, Ala64Ser, Asn70Ser, and Ser137Ala) have evolved substitutions in parallel and exhibit significant d(N)/d(S) >1. Homology modeling of the full-length Limenitis arthemis astyanax L opsin placed all four substitutions within the chromophore-binding pocket. Strikingly, the Ser137Ala substitution is in the same position as a site that in primates is responsible for a 5- to 7-nm blue spectral shift. Our data show that some of the same amino acid sites are under positive selection in the photopigments of both butterflies and primates, spanning an evolutionary distance >500 million years.

Isolation of microsatellite loci in the Capricorn silvereye, Zosterops lateralis chlorocephalus (Aves: Zosteropidae)

The Capricorn silvereye (Zosterops lateralis chlorocephalus) is ideally suited to investigating the genetic basis of body size evolution. We have isolated and characterized a set of microsatellite markers for this species. Seven out of 11 loci were polymorphic. The number of alleles detected ranged from two to five and observed heterozygosities between 0.12 and 0.67. One locus, ZL49, was found to be sex-linked. This moderate level of diversity is consistent with that expected in an isolated, island population.

Supramolecular selection in molecular alloys

Complexes of the type \[M(phen)3](PF6)2 (M = Ni(II), Fe(II), Ru(II) and phen = 1,10-phenanthroline) were found to co-crystallize to form molecular alloys (solid solutions of molecules) with general formula \[MAxMB1–x(phen)3](PF6)2·0.5H2O in which the relative concentrations of the metal complexes in the crystals closely match those in the crystallizing solution. Consequently, the composition of the co-crystals can be accurately predicted and controlled by modulating the relative concentrations of the metal complexes in the crystallizing solution. Although they are chemically and structurally similar, complexes of the type \[M(bipy)3](PF6)2 (M = Ni(II), Fe(II), Ru(II) and bipy = 2,2′-bipyridine) display markedly different behavior upon co-crystallization. In this case, the resulting co-crystals of general formula \[MAxMB1–x(bipy)3](PF6)2 have relative concentrations of the constituent complexes that are markedly different from the relative concentrations of the complexes initially present in the crystallizing solution. For example, when the nickel and iron complexes are co-crystallized from a solution containing a 50:50 ratio of each, the result is the formation of some crystals with a higher proportion of iron and others with a higher proportion of nickel. The relative concentrations of the metal complexes in the crystals can vary from those in the crystallizing solutions by as much as 15%. This result was observed for a range of combinations of metal complexes (Ni/Fe, Ni/Ru, and Fe/Ru) and a range of starting concentrations in the crystallizing solutions (90:10 through to 10:90 in 10% increments). To explain this remarkable result, we introduce the concept of “supramolecular selection”, which is a process driven by molecular recognition that leads to the partially selective aggregation of like molecules during crystallization.

Beyond barcoding : a mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117–200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.

Experimental observations of rapid maize streak virus evolution reveal a strand-specific nucleotide substitution bias

Background. Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ∼10-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered. Results. We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 × 10-4 and 7.9 × 10-4 subs/site/year. Conclusion. These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules. © 2008 Walt et al; licensee BioMed Central Ltd.

Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution

Background Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae), the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. Results Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots - of the breakpoints required to re-create MSV-MatA. Although the MSV-sensitive maize genotype gave rise to the greatest variety of recombinants, the measured fitness of each of these recombinants correlated with their similarity to MSV-MatA. Conclusions The mechanistic predispositions of different MSV genomic regions to recombination can strongly influence the accessibility of high-fitness MSV recombinants. The frequency with which the fittest recombinant MSV genomes arise also correlates directly with the escalating selection pressures imposed by increasingly MSV-resistant maize hosts.