Antimicrobial potential of the ionophore monensin on freshwater biofilm bacteria.

Microorganisms play key roles in stream ecosystems, but comparatively little is known about the resilience of freshwater bacterial communities and their susceptibility to the chemical by-products of agricultural land use. Antibiotics used in the agricultural sector are of particular concern and have been detected in waterways associated with agricultural land. Despite widespread agricultural intensification globally and the sector's high antibiotic use, the effects of agricultural antibiotic by-products on stream microbial communities have yet to be characterised. We investigated the impacts of the antibiotic monensin on microbial biofilm communities in a simulated contamination event using streamside-replicated channels. A 24-h pulse experiment in flow channels precolonised by stream biofilm microbial communities contrasted the effects of monensin concentrations ranging from realistic to extreme toxicity levels (1-550 ug L(-1)). Biofilm community composition was characterised immediately before and after the pulse for several weeks using automated ribosomal intergenic spacer analysis. Despite applying acutely toxic levels of monensin, only limited effects to biofilm community composition were detected immediately after antibiotic application, and these disappeared within 4 days. Rather, temporal factors drove biofilm differences, highlighting the overriding importance of wider, catchment-level, physiochemical hydrological influences on structuring freshwater biofilm communities, as opposed to localised and sporadic agricultural surface runoff contamination events containing antibiotics.

Antibiotic resistance genes in freshwater biofilms along a whole river.

A key problem challenging public health officials' efforts to stem the spread of antibiotic resistance is the potential increase of resistance in the environment. Yet, despite recent and significant changes to agricultural land in New Zealand, as well as the sector's high antibiotic use, the influence on antibiotic resistance in the environment remained uncharacterised. Spatial and temporal dynamics of antibiotic resistance genes in freshwater biofilms from NZ's fourth longest river as it transitioned between low and high intensity farming were examined for 1 year. Polymerase chain reaction was employed to gauge the level of resistance present. Biofilms were screened for 10 genes conferring resistance to antibiotics used in humans only and both humans and agricultural animals. Three genes were detected, one which conferred resistance to the important human-only use antibiotic vancomycin. Detected at the two downstream sites only, and those subject to the highest combined land-use stressors, the three genes indicated an elevated presence of antibiotic resistance in relation to surrounding land use; 7.7% versus 2% across the whole river system. The detection of a gene conferring resistance to an important human-only use antibiotic was particularly concerning and highlighted human-based contamination sources along the river, in addition to those of agricultural origin.

Land-use change and emerging public health risks in New Zealand: assessing Giardia risks.

Agriculture is key to New Zealand's economy with land-use conversions in response to market forces occurring regularly, like that of recent dairy intensification throughout the country. However, land-use conversion can occasionally result in unexpected and significant consequences for public health that need to be accurately estimated and subsequently managed accordingly. For example, dairy cattle have high Giardia prevalence in New Zealand and identical strains from infected humans and cattle located in the same geographical region have recently been reported. Thus, the high rates of human infections in New Zealand compared to similar socioeconomic countries caused by the waterborne pathogen Giardia are particularly concerning given the increasing dairy cattle populations on the landscape. However, the ability of traditional, evidence-based, epidemiological approaches to detect such causal relationships between land-use and Giardia infections is limited given the many possible indirect links between the two, in turn highlighting the need to develop appropriate risk assessment techniques. As such, the general requirements for and development of risk assessment frameworks to evaluate the likelihood of public health risks from waterborne pathogens are introduced and explored using Giardia in New Zealand as an example. Specifically, the importance of recent advances in Giardia-based knowledge, the incorporation of such data into existing risk assessment frameworks and the influence of remaining research gaps are each discussed for expanding currently available risk assessment tools. Not surprisingly, the availability of appropriate risk assessment tools for agencies responsible for public health and environmental management would ensure the public health risks for Giardia resulting from land-use change could be quantified holistically and strategies subsequently developed through active agency communication to minimise such risks.

Using native riparian barriers to reduce Giardia in agricultural runoff to freshwater ecosystems.

Waterway degradation in agricultural settings is caused by direct and diffuse sources of pollution. Waterway fencing focuses on reducing direct faecal contamination, but the extent to which it reduces overland surface runoff of pathogens is unknown. This study evaluated the potential of four riparian treatments to reduce Giardia in saturation excess surface runoff entering the waterway. Treatment 1 comprised exotic pasture grass and weeds that regenerated from bare soil between the fence and the waterway in the absence of cattle grazing and was compared with three others comprising monocultural plantings of New Zealand native grassland plants. Runoff experiments involving Giardia were performed after planting, both prior to and following the summer growing season. Giardia was not detected from any plot prior to cyst addition. In spring the native 'C. secta', 'A. lessoniana' and 'C. richardii' treatments showed significantly greater reductions in Giardia in runoff than the 'exotic grasses' treatment, while in autumn the 'C. richardii' treatment reduced Giardia more than the 'exotic grasses/weeds'. A reduction in public health risk should follow from riparian vegetation, whether exotic or native, but with an added benefit in the case of the native tussock grass C. richardii, due to the associated lower runoff rate.

Recently planted vegetation strips reduce Giardia runoff reaching waterways.

Current methods for tracking pathogens across farmland and into surrounding waterways via runoff are limited and typically have been developed using artificially created landscapes. No studies have investigated how Giardia in farm runoff moves across the landscape, despite high prevalence rates in dairy cattle (Bos taurus) worldwide. Here, we report the development of a field-based tracking method specific for Giardia movement in runoff and use this technique to compare the pathogen reduction capability of recently planted vegetation strips with bare soil strips cleared of vegetation. Such scenarios represent typical events in schemes to plant vegetation barriers aimed at reducing waterway contamination. A significant treatment effect was identified, with 26% fewer Giardia detected in runoff collected from the planted strip (P = 0.006). These results highlight the immediate benefit of pathogen removal to be gained from vegetation planting. The successful discrimination of treatment effects by this new technique will enable the assessment of different vegetation types on runoff reduction and the effects of plant development over time.

Molecular characterization of Giardia isolates from calves and humans in a region in which dairy farming has recently intensified.

Giardiasis is a notifiable disease of high prevalence in New Zealand, but there is limited knowledge about the sources of Giardia duodenalis genotypes that can potentially cause human infections. Dairy calves are one environmental source of Giardia isolates, but it is unknown whether they harbor genotypes that are potentially capable of causing infections in humans. To address these questions, 40 Giardia isolates from calves and 30 from humans, living in the same region and collected over a similar period, were genotyped using the beta-giardin gene. The G. duodenalis genetic assemblages A and B were identified from both calves and humans, and genotype comparisons revealed a substantial overlap of identical genotypes from the two hosts for both assemblages. Significantly, no assemblage E (the genotype commonly found in cattle elsewhere in the world) has been detected in New Zealand livestock to date. Given recent and rapid land use conversions to dairy farming in many South Island regions of New Zealand, an increasingly large concentration of domestic cattle harboring genotypes potentially capable of causing infections in humans is particularly concerning.

Tests of parallel molecular evolution in a long-term experiment with Escherichia coli.

The repeatability of evolutionary change is difficult to quantify because only a single outcome can usually be observed for any precise set of circumstances. In this study, however, we have quantified the frequency of parallel and divergent genetic changes in 12 initially identical populations of Escherichia coli that evolved in identical environments for 20,000 cell generations. Unlike previous analyses in which candidate genes were identified based on parallel phenotypic changes, here we sequenced four loci (pykF, nadR, pbpA-rodA, and hokB/sokB) in which mutations of unknown effect had been discovered in one population, and then we compared the substitution pattern in these "blind" candidate genes with the pattern found in 36 randomly chosen genes. Two candidate genes, pykF and nadR, had substitutions in all 11 other populations, and the other 2 in several populations. There were very few cases, however, in which the exact same mutations were substituted, in contrast to the findings from conceptually related work performed with evolving virus populations. No random genes had any substitutions except in four populations that evolved defects in DNA repair. Tests of four different statistical aspects of the pattern of molecular evolution all indicate that adaptation by natural selection drove the parallel changes in these candidate genes.

Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates / Divergencia genómica de algunas cepas de Escherichia coli: pruebas de la transferencia horizontal y variación en las velocidades de mutación

This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago (AU)

Se secuenciaron 36 regiones del genoma de Escherichia coli B elegidas al azar y que están presentes en la mayoría o en todos los genomas de E. coli secuenciados. Se examinaron las relaciones filogenéticas entre cepas de E. coli y se buscaron pruebas de transferencia génica horizontal y de variación en la tasa de mutación. El árbol filogenético conjunto de genes indica que E. coli B y K-12 son las cepas con un parentesco más estrecho, mientras que E. coli O157:H7 se encuentra más alejada y aún más lo están Shigella flexneri 2a y E. coli CFT073, siendo esta última la más distante de todas. En el grupo B, K-12 y O157:H7, varias regiones indican que hay topologías alternativas. La transferencia génica horizontal es una explicación plausible de estas incongruencias filogenéticas, pero también hemos hallado pruebas de una evolución más rápida en sitios sinónimos en el linaje O157:H7. Así pues, una interpretación más profunda de estos resultados queda confundida por una asociación entre unos genes que muestran una evolución más rápida y otros que son transferidos horizontalmente. Usando genes que apoyan los grupos B y K-1, y empleando una estima de la tasa de mutación obtenida a partir de un experimento de evolución a largo plazo con E. coli B y suponiendo 200 generaciones por año, se estimó que las cepas B y K-12 divergieron hace varios cientos de miles de años, mientras que O157:H7 se separó de su ancestro común hace entre 1,5 y 2 millones de años (AU)

Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates.

This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago.

Rates of DNA sequence evolution in experimental populations of Escherichia coli during 20,000 generations.

We examined rates of DNA sequence evolution in 12 populations of Escherichia coli propagated in a glucose minimal medium for 20,000 generations. Previous work saw mutations mediated by mobile elements in these populations, but the extent of other genomic changes was not investigated. Four of the populations evolved defects in DNA repair and became mutators. Some 500 bp was sequenced in each of 36 genes for 50 clones, including 2 ancestral variants, 2 clones from each population at generation 10,000, and 2 from each at generation 20,000. Ten mutations were found in total, all point mutations including mostly synonymous substitutions and nonsynonymous polymorphisms; all 10 were found in mutator populations. We compared the observed sequence evolution to predictions based on different scenarios. The number of synonymous substitutions is lower than predicted from measured mutation rates in E. coli, but the number is higher than rates based on comparing E. coli and Salmonella genomes. Extrapolating to the entire genome, these data predict about 250 synonymous substitutions on average per mutator population, but only about 3 synonymous substitutions per nonmutator population, during 20,000 generations. These data illustrate the challenge of finding sequence variation among bacterial isolates that share such a recent ancestor. However, this limited variation also provides a useful baseline for research aimed at finding the beneficial substitutions in these populations.