Inactivation of the antibacterial and cytotoxic properties of silver ions by biologically relevant compounds

There has been a recent surge in the use of silver as an antimicrobial agent in a wide range of domestic and clinical products, intended to prevent or treat bacterial infections and reduce bacterial colonization of surfaces. It has been reported that the antibacterial and cytotoxic properties of silver are affected by the assay conditions, particularly the type of growth media used in vitro. The toxicity of Ag+ to bacterial cells is comparable to that of human cells. We demonstrate that biologically relevant compounds such as glutathione, cysteine and human blood components significantly reduce the toxicity of silver ions to clinically relevant pathogenic bacteria and primary human dermal fibroblasts (skin cells). Bacteria are able to grow normally in the presence of silver nitrate at >20-fold the minimum inhibitory concentration (MIC) if Ag+ and thiols are added in a 1:1 ratio because the reaction of Ag+ with extracellular thiols prevents silver ions from interacting with cells. Extracellular thiols and human serum also significantly reduce the antimicrobial activity of silver wound dressings Aquacel-Ag (Convatec) and Acticoat (Smith & Nephew) to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli in vitro. These results have important implications for the deployment of silver as an antimicrobial agent in environments exposed to biological tissue or secretions. Significant amounts of money and effort have been directed at the development of silver-coated medical devices (e.g. dressings, catheters, implants). We believe our findings are essential for the effective design and testing of antimicrobial silver coatings

Transcript abundance of photorhabdus insect-related (Pir) toxin in Manduca sexta and Galleria mellonella infections

In this study, we assessed pirAB toxin transcription in Photorhabdus luminescens laumondii (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: Galleria mellonella and Manduca sexta. Three portals of entry were utilized for the forced infection assays: (a) integument; (b) the digestive route (via mouth and anus); and (c) the tracheal route (via spiracles). We also assessed plu4093-2 transcription during the course of a natural infection; this is when the bacteria are delivered by Heterorhabditis bacteriophora nematodes. Transcript abundance in G. mellonella was higher than in M. sexta at two of the observed time points: 15 and 18 h. Expression of pirAB plu4093-2 reached above endogenous control levels at 22 h in G. mellonella but not in M. sexta. Overall, pirAB plu4093-2 transcripts were not as highly expressed in M. sexta as in G. mellonella, from 15 to 22 h. This is the first study to directly compare pirAB plu4093-2 toxin transcript production considering different portals of entry

Evolutionary rewiring of bacterial regulatory networks

Bacteria have evolved complex regulatory networks that enable integration of multiple intracellular and extracellular signals to coordinate responses to environmental changes. However, our knowledge of how regulatory systems function and evolve is still relatively limited. There is often extensive homology between components of different networks, due to past cycles of gene duplication, divergence, and horizontal gene transfer, raising the possibility of cross-talk or redundancy. Consequently, evolutionary resilience is built into gene networks – homology between regulators can potentially allow rapid rescue of lost regulatory function across distant regions of the genome. In our recent study [Taylor, et al. Science (2015), 347(6225)] we find that mutations that facilitate cross-talk between pathways can contribute to gene network evolution, but that such mutations come with severe pleiotropic costs. Arising from this work are a number of questions surrounding how this phenomenon occurs

Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system

A central process in evolution is the recruitment of genes to regulatory networks. We engineered immotile strains of the bacterium Pseudomonas fluorescens that lack flagella due to deletion of the regulatory gene fleQ. Under strong selection for motility, these bacteria consistently regained flagella within 96 hours via a two-step evolutionary pathway. Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant homologue of FleQ, which begins to commandeer control of the fleQ regulon at the cost of disrupting nitrogen uptake and assimilation. Step 2 is a switch-of-function mutation that redirects NtrC away from nitrogen uptake and towards its novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps

From insect to man: Photorhabdus sheds light on the emergence of human pathogenicity

Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called “nutritional virulence” strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway

Temperature restriction in entomopathogenic bacteria

Temperature plays an important role in bacteria-host interactions and can be a determining factor for host switching. In this study we sought to investigate the reasons behind growth temperature restriction in the entomopathogenic enterobacterium Photorhabdus. Photorhabdus has a complex dual symbiotic and pathogenic life cycle. The genus consists of 19 species but only one subgroup, previously all classed together as Photorhabdus asymbiotica, have been shown to cause human disease. These clinical isolates necessarily need to be able to grow at 37°C, whilst the remaining species are largely restricted to growth temperatures below 34°C and are therefore unable to infect mammalian hosts. Here, we have isolated spontaneous mutant lines of Photorhabdus laumondii DJC that were able to grow up to 36–37°C. Following whole genome sequencing of 29 of these mutants we identified a single gene, encoding a protein with a RecG-like helicase domain that for the majority of isolates contained single nucleotide polymorphisms. Importantly, provision of the wild-type allele of this gene in trans restored the temperature restriction, confirming the mutations are recessive, and the dominant effect of the protein product of this gene. The gene appears to be part of a short three cistron operon, which we have termed the Temperature Restricting Locus (TRL). Transcription reporter strains revealed that this operon is induced upon the switch from 30 to 36°C, leading to replication arrest of the bacteria. TRL is absent from all of the human pathogenic species so far examined, although its presence is not uniform in different strains of the Photorhabdus luminescens subgroup. In a wider context, the presence of this gene is not limited to Photorhabdus, being found in phylogenetically diverse proteobacteria. We therefore suggest that this system may play a more fundamental role in temperature restriction in diverse species, relating to as yet cryptic aspects of their ecological niches and life cycle requirements

Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages

Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonas tolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonas agarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance

A thin film detection/response system for pathogenic bacteria

This paper describes the modification of nonwoven fabric such that it responds by releasing an encapsulated antimicrobial from within an attached vesicle in response to two species of pathogenic bacteria (Staphylococcus aureus MSSA 476 and Pseudomonas aeruginosa PAO1), but does not respond to nonpathogenic Escherichia coli DH5α. This concept is based on the generalization that a majority of pathogenic bacteria secrete virulence factors such as toxins and lipases that actively damage cell membranes, typically observed as tissue damage around infected wounds, while nonpathogenic bacteria do not (or not at high concentration). The eventual aim of this work is to produce responsive dressings which release antimicrobials and change color only on infected wounds. This paper details preliminary approaches to achieving this goal, including vesicle−bacteria studies in aqueous suspension, and fluorescence imaging of fluorescein containing vesicles lysed by S. aureus and P. aeruginosa, but not by E. coli

The effect of silver nitrate on the growth of <i>Staphylococcus aureus</i> MSSA476 in different media.

<p>AgNO₃ was added to growth media at the indicated concentrations (µmol dm⁻³) in (A) LB; (B) LB + 50 mg/mL HSA; (C) LB + 50% human serum (<i>v/v</i>); (D) LB + 1 mmol dm⁻³ GSH. GSH, reduced glutathione; HSA, human serum albumin (the major blood protein). Error bars  =  SD, n = 3.</p