Exploring the parameters of post-segregational killing using heterologous expression of secreted toxin barnase and antitoxin barstar in an Escherichia coli case study.

Post-segregational killing (PSK) is a phenotype determined by plasmids using a toxin and an antitoxin gene pair. Loss of the genes depletes the cell's reserve of antitoxin and allows the toxin to act upon the cell. PSK benefits mobile elements when it increases reproductive success relative to other mobile competitors. A side effect of PSK is that plasmids become refractory to displacement from the cell during growth as a monoculture. Most PSK systems use a cytoplasmic toxin, but the external toxins of bacteriocins also have a PSK-like effect. It may be that any toxin and antitoxin gene pair can demonstrate PSK when it is on a plasmid. The secreted ribonuclease barnase and its protein inhibitor barstar have features in common with PSK modules, though their native context is chromosomal. We hypothesized that their recruitment to a plasmid could produce an emergent PSK phenotype. Others had shown that secreted barnase could exert a lethal effect on susceptible bacteria similarly to bacteriocins. However, barnase toxicity did not occur under the conditions tested, suggesting that barnase is toxic to neighbouring cells only under very specific conditions. Bacteriocins are only produced under some conditions, and some conditionality on toxin function or release may be advantageous in general to PSKs with external toxins because it would prevent killing of potential plasmid-naive hosts. Too much conditionality, however, would limit how advantageous the gene pair was to mobile elements, making the genes unlikely to be recruited as a PSK system.

Herbicide ingredients change Salmonella enterica sv. Typhimurium and Escherichia coli antibiotic responses.

Herbicides are frequently released into both rural and urban environments. Commercial herbicide formulations induce adaptive changes in the way bacteria respond to antibiotics. Salmonella enterica sv. Typhimurium and Escherichia coli were exposed to common co-formulants of formulations, and S. enterica sv. Typhimurium was exposed to active ingredients dicamba, 2,4-D and glyphosate to determine what ingredients of the commercial formulations caused this effect. Co-formulants Tween80 and carboxymethyl cellulose induced changes in response, but the pattern of the responses differed from the active ingredients, and effect sizes were smaller. A commercial wetting agent did not affect antibiotic responses. Active ingredients induced changes in antibiotic responses similar to those caused by complete formulations. This occurred at or below recommended application concentrations. Targeted deletion of efflux pump genes largely neutralized the adaptive response in the cases of increased survival in antibiotics, indicating that the biochemistry of induced resistance was the same for formulations and specific ingredients. We found that glyphosate, dicamba, and 2,4-D, as well as co-formulants in commercial herbicides, induced a change in susceptibility of the potentially pathogenic bacteria E. coli and S. enterica to multiple antibiotics. This was measured using the efficiency of plating (EOP), the relative survival of the bacteria when exposed to herbicide and antibiotic, or just antibiotic, compared to survival on permissive media. This work will help to inform the use of non-medicinal chemical agents that induce changes in antibiotic responses.

Why so narrow: Distribution of anti-sense regulated, type I toxin-antitoxin systems compared with type II and type III systems.

Toxin-antitoxin (TA) systems are gene modules that appear to be horizontally mobile across a wide range of prokaryotes. It has been proposed that type I TA systems, with an antisense RNA-antitoxin, are less mobile than other TAs that rely on direct toxin-antitoxin binding but no direct comparisons have been made. We searched for type I, II and III toxin families using iterative searches with profile hidden Markov models across phyla and replicons. The distribution of type I toxin families were comparatively narrow, but these patterns weakened with recently discovered families. We discuss how the function and phenotypes of TA systems as well as biases in our search methods may account for differences in their distribution.

Indigenous food sources as vectors of Escherichia coli and antibiotic resistance.

The contamination of surface waters by fecal bacteria, measured by the number of Escherichia coli, is a significant public health issue. When these bacteria are also resistant to antimicrobials, infections are more complicated to treat. While water is regularly tested at recreational sites, wild-harvested foods, known as mahinga kai by the indigenous Maori people of Aotearoa New Zealand, are commonly overlooked as a source of exposure to potential pathogens and antimicrobial resistance (AMR). We investigate two likely sources of risk from harvesting aquatic wild foods. The first is water contact, and the second is contact with/ingestion of the harvest. We used E. coli as a proxy for microbial water quality at harvesting sites. Two popular mahinga kai species were also harvested and assessed. We found antibiotic-resistant bacteria on watercress (Nasturtium officinale) and cockles (Austrovenus stutchburyi). One-third of E. coli isolates were conjugative donors of at least one resistance phenotype. Tank experiments were used to track the internalization of E. coli by Greenshell/lip mussels (Perna canaliculus). Greenshell mussels kept at environmentally relevant concentrations of E. coli were colonized to levels considered unsafe for human consumption in 24 h. Finally, we measured horizontal gene transfer between bacteria within the shellfish, what we termed 'intra-shellular' conjugation. The transmission frequency of plasmid RP4 was significantly higher in mussels than in water alone. Our results indicate that shellfish could promote the dissemination of antibiotic resistance. They highlight the need to limit or reduce human pathogenic bacteria where food is gathered.

The coevolution of toxin and antitoxin genes drives the dynamics of bacterial addiction complexes and intragenomic conflict.

Bacterial genomes commonly contain 'addiction' gene complexes that code for both a toxin and a corresponding antitoxin. As long as both genes are expressed, cells carrying the complex can remain healthy. However, loss of the complex (including segregational loss in daughter cells) can entail death of the cell. We develop a theoretical model to explore a number of evolutionary puzzles posed by toxin-antitoxin (TA) population biology. We first extend earlier results demonstrating that TA complexes can spread on plasmids, as an adaptation to plasmid competition in spatially structured environments, and highlight the role of kin selection. We then considered the emergence of TA complexes on plasmids from previously unlinked toxin and antitoxin genes. We find that one of these traits must offer at least initially a direct advantage in some but not all environments encountered by the evolving plasmid population. Finally, our study predicts non-transitive 'rock-paper-scissors' dynamics to be a feature of intragenomic conflict mediated by TA complexes. Intragenomic conflict could be sufficient to select deleterious genes on chromosomes and helps to explain the previously perplexing observation that many TA genes are found on bacterial chromosomes.

Isolation and characterization of bacteriophages infecting Xanthomonas arboricola pv. juglandis, the causal agent of walnut blight disease.

Walnut orchards suffer from a blight caused by the bacteria Xanthomonas arboricola pv. juglandis. These bacteria can be infected by viral bacteriophages and this study was carried out to isolate and characterize bacteriophages from walnut orchards located throughout the South Island of New Zealand. Twenty six X. arboricola phages were isolated from three hundred and twenty six samples of plant material representing phyllosphere and rhizosphere ecosystems. The phage isolates were characterized by host-range, plaque and particle morphology, restriction digest and phylogenetic analysis and stability under various storage conditions. From capsid and tail dimensions the bacteriophages were considered to belong to the double-stranded DNA families Podoviridae and Siphoviridae. Of the twenty six bacteriophages, sixteen belonged to Podoviridae and were found both in the phyllosphere and rhizosphere. In contrast, Siphoviridae were present only in the rhizosphere isolates. Phage genome sizes ranged from 38.0 to 52.0 kb from a Hind III restriction digestion and had in common a 400 kb fragment that was identical at the DNA level. Despite the similar restriction patterns, maximum parsimony bootstrap analysis showed that the phage were members of different groups. Finally, we hypothesise that these phage might have use in a biocontrol strategy and therefore storage stability and efficacy was tested. Titres declined more than 50% over a 12-months storage period. Deep-freezing temperatures (-34°C) increased while chloroform decreased the stability.

Human lactoferrin increases Helicobacter pylori internalisation into AGS cells.

Helicobacter pylori has high global infection rates and can cause other undesirable clinical manifestations such as duodenal ulcer (DU) and gastric cancer (GC). Frequencies of re-infection after therapeutic clearance and rates of DU versus GC vary geographically and differ markedly between developed and developing countries, which suggests additional factors may be involved. The possibility that, in vivo, lactoferrin (Lf) may play a subtle role in modulating micronutrient availability or bacterial internalisation with implications for disease etiology is considered. Lf is an iron binding protein produced in mammals that has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonise mammalian hosts have adapted to living in high Lf environments and we investigated if this included the gastric pathogen H. pylori. We found that H. pylori was able to use iron from fully iron-saturated human Lf (hLf) whereas partially iron-saturated hLf (apo) did not increase H. pylori growth. Instead, apo-hLf increased adherence to and internalisation of bacteria into cultured epithelial cells. By increasing internalisation, we speculate that apo-human lactoferrin may contribute to H. pylori's ability to persistence in the human stomach, an observation that potentially has implications for the risk of H. pylori-associated disease.

Draft Genome Sequences of 15 Multidrug-Resistant Escherichia coli Strains Isolated from Indigenous Foods and Food-Gathering Sites in Aotearoa, New Zealand.

We report the draft genomes of 15 multidrug-resistant and potentially pathogenic Escherichia coli strains isolated from watercress, cockles, or the surrounding water in Aotearoa, New Zealand.

Copper and nanostructured anatase rutile and carbon coatings induce adaptive antibiotic resistance.

Contaminated surfaces are vehicles for the spread of infectious disease-causing microorganisms. A strategy to prevent their spread is applying antimicrobial coatings to surfaces. Both nanostructured anatase rutile and carbon (NsARC), a TiO2 formulation, and copper are examples of antimicrobial agents that are used in making or coating door handles and similar surfaces, to reduce microbial loads. Antimicrobial surfaces have been extensively tested for antimicrobial activity but not sublethal effects, such as exposure-associated multiple antibiotic resistance phenotypes usually caused by induction of efflux pump genes. The possibility of NsARC and copper inducing indicative efflux pump pathways was investigated by monitoring the expression of mScarlet fluorescent protein (FP) in two reporter strains of Escherichia coli. There was an increase in the expression of FP in the reporter strains exposed to NsARC and copper relative to the inert control composed of stainless steel. Furthermore we tested E. coli and Staphylococcus aureus following 8 h of exposure to NsARC for changes in resistance to selected antibiotics. E. coli that were exposed to NsARC became more susceptible to kanamycin but there was no significant change in susceptibility of S. aureus to any tested antibiotics. These findings suggests that even though NsARC and copper are antimicrobial, they also have some potential to cause unintended phenotypes.

Are null segregants new combinations of heritable material and should they be regulated?

Through genome editing and other techniques of gene technology, it is possible to create a class of organism called null segregants. These genetically modified organisms (GMOs) are products of gene technology but are argued to have no lingering vestige of the technology after the segregation of chromosomes or deletion of insertions. From that viewpoint regulations are redundant because any unique potential for the use of gene technology to cause harm has also been removed. We tackle this question of international interest by reviewing the early history of the purpose of gene technology regulation. The active ingredients of techniques used for guided mutagenesis, e.g., site-directed nucleases, such as CRISPR/Cas, are promoted for having a lower potential per reaction to create a hazard. However, others see this as a desirable industrial property of the reagents that will lead to genome editing being used more and nullifying the promised hazard mitigation. The contest between views revolves around whether regulations could alter the risks in the responsible use of gene technology. We conclude that gene technology, even when used to make null segregants, has characteristics that make regulation a reasonable option for mitigating potential harm. Those characteristics are that it allows people to create more harm faster, even if it creates benefits as well; the potential for harm increases with increased use of the technique, but safety does not; and regulations can control harm scaling.

Antimicrobial and biofilm-disrupting nanostructured TiO2 coating demonstrating photoactivity and dark activity.

Antimicrobial materials are tools used to reduce the transmission of infectious microorganisms. Photo-illuminated titania (TiO2) is a known antimicrobial material. Used as a coating on door handles and similar surfaces, it may reduce viability and colonization by pathogens and limit their spread. We tested the survival of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Saccharomyces cerevisiae on a nano-structured TiO2-based thin film, called 'NsARC', and on stainless steel under a variety of light wavelengths and intensities. There was significantly less survival (P <0.001) of all the organisms tested on NsARC compared to inert uncoated stainless steel under all conditions. NsARC was active in the dark and possible mechanisms for this are suggested. NsARC inhibited biofilm formation as confirmed by scanning electron microscopy. These results suggest that NsARC can be used as a self-cleaning and self-sterilizing antimicrobial surface coating for the prevention and reduction in the spread of potentially infectious microbes.

Within-host competition selects for plasmid-encoded toxin-antitoxin systems.

Toxin-antitoxin (TA) systems are commonly found on bacterial plasmids. The antitoxin inhibits toxin activity unless the system is lost from the cell. Then the shorter lived antitoxin degrades and the cell becomes susceptible to the toxin. Selection for plasmid-encoded TA systems was initially thought to result from their reducing the number of plasmid-free cells arising during growth in monoculture. However, modelling and experiments have shown that this mechanism can only explain the success of plasmid TA systems under a restricted set of conditions. Previously, we have proposed and tested an alternative model explaining the success of plasmid TA systems as a consequence of competition occurring between plasmids during co-infection of bacterial hosts. Here, we test a further prediction of this model, that competition between plasmids will lead to the biased accumulation of TA systems on plasmids relative to chromosomes. Transposon-encoded TA systems were added to populations of plasmid-containing cells, such that TA systems could insert into either plasmids or chromosomes. These populations were enriched for transposon-containing cells and then incubated in environments that did, or did not, allow effective within-host plasmid competition to occur. Changes in the ratio of plasmid- to chromosome-encoded TA systems were monitored. In agreement with our model, we found that plasmid-encoded TA systems had a competitive advantage, but only when host cells were sensitive to the effect of TA systems. This result demonstrates that within-host competition between plasmids can select for TA systems.

Should dsRNA treatments applied in outdoor environments be regulated?

The New Zealand Environmental Protection Authority (EPA) issued a Decision that makes the use of externally applied double-stranded (ds)RNA molecules on eukaryotic cells or organisms technically out of scope of legislation on new organisms, making risk assessments of such treatments in the open environment unnecessary. The Decision was based on its view that the treatment does not create new or genetically modified organisms and rests on the EPA's conclusions that dsRNA is not heritable and is not a mutagen. For these reasons EPA decided that treatments using dsRNA do not modify genes or other genetic material. I found from an independent review of the literature on the topic indicated, however, that each of the major scientific justifications relied upon by the EPA was based on either an inaccurate interpretation of evidence or failure to consult the research literature pertaining to additional types of eukaryotes. The Decision also did not take into account the unknown and unique eukaryotic biodiversity of New Zealand. The safe use of RNA-based technology holds promise for addressing complex and persistent challenges in public health, agriculture and conservation. However, by failing to restrict the source or means of modifying the dsRNA, the EPA removed regulatory oversight that could prevent unintended consequences of this new technology such as suppression of genes other than those selected for suppression or the release of viral genes or genomes by failing to restrict the source or means of modifying the dsRNA.

Effects of sub-lethal concentrations of copper ammonium acetate, pyrethrins and atrazine on the response of Escherichia coli to antibiotics.

Background: Antibiotic resistance in human and animal pathogens is mainly the outcome of human use of antibiotics. However, bacteria are also exposed to thousands of other antimicrobial agents. Increasingly those exposures are being investigated as co-selective agents behind the rapid rise and spread of resistance in bacterial pathogens of people and our domesticated animals. Methods: We measured the sub-lethal effects on antibiotic tolerance of the human pathogen/commensal Escherichia coli caused by exposure to three common biocide formulations based on either copper, pyrethrins, or atrazine as active ingredients. The influence of the efflux pump AcrAB-TolC was investigated using deletion strains, and the persistence of observed effects was determined. Results: Some effects were seen for all biocides, but the largest effects were observed with copper in combination with the antibiotic tetracycline. The effect was caused by both the induction of the adaptive efflux system and by chelation of the antibiotic by copper. Finally, persistence of the adaptive response was measured and found to persist for about two generations. Conclusions: Through a combination of microbe-chemical and chemical-chemical interactions, humanity may be creating micro-environments in which resistance evolution is accelerated.

Prevalence of antibiotic-resistant Escherichia coli isolated from urban and agricultural streams in Canterbury, New Zealand.

Baseline studies are needed to identify environmental reservoirs of non-pathogenic but associating microbiota or pathogenic bacteria that are resistant to antibiotics and to inform safe use of freshwater ecosystems in urban and agricultural settings. Mesophilic bacteria and Escherichia coli were quantified and isolated from water and sediments of two rivers, one in an urban and one in an agricultural area near Christchurch, New Zealand. Resistance of E. coli to one or more of nine different antibiotics was determined. Additionally, selected strains were tested for conjugative transfer of resistances. Despite having similar concentrations of mesophilic bacteria and E. coli, the rivers differed in numbers of antibiotic-resistant E. coli isolates. Fully antibiotic-susceptible and -resistant strains coexist in the two freshwater ecosystems. This study was the first phase of antibiotic resistance profiling in an urban setting and an intensifying dairy agroecosystem. Antibiotic-resistant E. coli may pose different ingestion and contact risks than do susceptible E. coli. This difference cannot be seen in population counts alone. This is an important finding for human health assessments of freshwater systems, particularly where recreational uses occur downstream.

Nanostructured TiO2 anatase-rutile-carbon solid coating with visible light antimicrobial activity.

TiO2 photocatalyst is of interest for antimicrobial coatings on hospital touch-surfaces. Recent research has focused on visible spectrum enhancement of photocatalytic activity. Here, we report TiO2 with a high degree of nanostructure, deposited on stainless steel as a solid layer more than 10 µm thick by pulsed-pressure-MOCVD. The TiO2 coating exhibits a rarely-reported microstructure comprising anatase and rutile in a composite with amorphous carbon. Columnar anatase single crystals are segmented into 15-20 nm thick plates, resulting in a mille-feuilles nanostructure. Polycrystalline rutile columns exhibit dendrite generation resembling pine tree strobili. We propose that high growth rate and co-deposition of carbon contribute to formation of the unique nanostructures. High vapor flux produces step-edge instabilities in the TiO2, and solid carbon preferentially co-deposits on certain high energy facets. The equivalent effective surface area of the nanostructured coating is estimated to be 100 times higher than standard TiO2 coatings and powders. The coatings prepared on stainless steel showed greater than 3-log reduction in viable E coli after 4 hours visible light exposure. The pp-MOCVD approach could represent an up-scalable manufacturing route for supported catalysts of functional nanostructured materials without having to make nanoparticles.

Agrichemicals and antibiotics in combination increase antibiotic resistance evolution.

Antibiotic resistance in our pathogens is medicine's climate change: caused by human activity, and resulting in more extreme outcomes. Resistance emerges in microbial populations when antibiotics act on phenotypic variance within the population. This can arise from either genotypic diversity (resulting from a mutation or horizontal gene transfer), or from differences in gene expression due to environmental variation, referred to as adaptive resistance. Adaptive changes can increase fitness allowing bacteria to survive at higher concentrations of antibiotics. They can also decrease fitness, potentially leading to selection for antibiotic resistance at lower concentrations. There are opportunities for other environmental stressors to promote antibiotic resistance in ways that are hard to predict using conventional assays. Exploiting our previous observation that commonly used herbicides can increase or decrease the minimum inhibitory concentration (MIC) of different antibiotics, we provide the first comprehensive test of the hypothesis that the rate of antibiotic resistance evolution under specified conditions can increase, regardless of whether a herbicide increases or decreases the antibiotic MIC. Short term evolution experiments were used for various herbicide and antibiotic combinations. We found conditions where acquired resistance arises more frequently regardless of whether the exogenous non-antibiotic agent increased or decreased antibiotic effectiveness. This is attributed to the effect of the herbicide on either MIC or the minimum selective concentration (MSC) of a paired antibiotic. The MSC is the lowest concentration of antibiotic at which the fitness of individuals varies because of the antibiotic, and is lower than MIC. Our results suggest that additional environmental factors influencing competition between bacteria could enhance the ability of antibiotics to select antibiotic resistance. Our work demonstrates that bacteria may acquire antibiotic resistance in the environment at rates substantially faster than predicted from laboratory conditions.

Prevalence and numbers of coliphages and Campylobacter jejuni bacteriophages in New Zealand foods.

Vegetable samples were tested for the presence of coliphages. None of the 55 samples contained these phages at concentrations greater than 10 g(-1) (the limit of detection). Spiking and recovery experiments indicated that the method was efficient at detecting coliphage T4 added to the food, and so it was concluded that phage titres were not being falsely underestimated. In addition 51 samples of chicken skin from retail portions were tested for the presence and numbers of coliphages and for presence only of Campylobacter jejuni phages. Coliphages were isolated from 46 samples (90.2% positive), at up to 2570 PFU 10 g sample(-1) but no C. jejuni phages were isolated. Several other methods were used to isolate C. jejuni phages from retail chicken but none was successful. However, when pooled whole chicken rinses from 39 flocks were tested for the presence of C. jejuni phages, 11 (28.2%) of the flocks were positive. It is possible that phages present on birds at the start of processing were either inactivated or simply diluted out during spin chilling. These data add to the body of information indicating that phages can readily be isolated from certain foods and indicate that consumers are exposed to them on a regular basis.

Problems in monitoring horizontal gene transfer in field trials of transgenic plants.

Transgenic crops are approved for release in some countries, while many more countries are wrestling with the issue of how to conduct risk assessments. Controls on field trials often include monitoring of horizontal gene transfer (HGT) from crops to surrounding soil microorganisms. Our analysis of antibiotic-resistant bacteria and of the sensitivity of current techniques for monitoring HGT from transgenic plants to soil microorganisms has two major implications for field trial assessments of transgenic crops: first, HGT from transgenic plants to microbes could still have an environmental impact at a frequency approximately a trillion times lower than the current risk assessment literature estimates the frequency to be; and second, current methods of environmental sampling to capture genes or traits in a recombinant are too insensitive for monitoring evolution by HGT. A model for HGT involving iterative short-patch events explains how HGT can occur at high frequencies but be detected at extremely low frequencies.