Dispersal of human and plant pathogens biofilms via nitric oxide donors at 4°C

Recent studies suggest that nitric oxide donors capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of biofilms. Encased in extracellular polymeric substances, human and plant pathogens within biofilms are significantly more resistant to sanitizers. This is particularly a problem in refrigerated environments where food is processed. In an exercise aimed to study the potential of nitric oxide donors as biofilm dispersal in refrigerated conditions, we compared the ability of different nitric oxide donors (SNAP, NO-aspirin and Noc-5) to dislodge biofilms formed by foodborne, human and plant pathogens treated at 4 °C. The donors SNAP and Noc-5 were efficient in dispersing biofilms formed by Salmonella enterica, pathogenic Escherichia coli and Listeria innocua. The biomasses were decreased up to 30 % when compared with the untreated controls. When the plant pathogens Pectobacterium sp. and Xanthomonas sp. were tested the dispersion was mainly limited to Pectobacterium carotovorum biofilms, decreasing up to 15 % after exposure to molsidomine. Finally, the association of selected nitric oxide donors with sanitizers (DiQuat, H2O2, peracetic acid and PhenoTek II) was effective in dispersing biofilms. The best dispersal was achieved by pre-treating P. carotovorum with molsidomine and then peracetic acid. The synergistic effect was estimated up to ~35 % in dispersal when compared with peracetic acid alone. The association of nitric oxide donors with sanitizers could provide a foundation for an improved sanitization procedure for cleaning refrigerate environments

Mosaic vs. Single Image Analysis with Confocal Microscopy of the Corneal Nerve Plexus for Diagnosis of Early Diabetic Peripheral Neuropathy

The assessment of the corneal nerve fibre plexus with corneal confocal microscopy (CCM) is an upcoming but still experimental method in the diagnosis of early stage diabetic peripheral neuropathy (DPN). Using an innovative imaging technique—Heidelberg Retina Tomograph equipped with the Rostock Cornea Module (HRT-RCM) and EyeGuidance module (EG)—we were able to look at greater areas of subbasal nerve plexus (SNP) in order to increase the diagnostic accuracy. The aim of our study was to evaluate the usefulness of EG instead of single image analysis in diagnosis of early stage DPN

Dispersal of human and plant pathogens biofilms via nitric oxide donors at 4°C

Recent studies suggest that nitric oxide donors capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of biofilms. Encased in extracellular polymeric substances, human and plant pathogens within biofilms are significantly more resistant to sanitizers. This is particularly a problem in refrigerated environments where food is processed. In an exercise aimed to study the potential of nitric oxide donors as biofilm dispersal in refrigerated conditions, we compared the ability of different nitric oxide donors (SNAP, NO-aspirin and Noc-5) to dislodge biofilms formed by foodborne, human and plant pathogens treated at 4 °C. The donors SNAP and Noc-5 were efficient in dispersing biofilms formed by Salmonella enterica, pathogenic Escherichia coli and Listeria innocua. The biomasses were decreased up to 30 % when compared with the untreated controls. When the plant pathogens Pectobacterium sp. and Xanthomonas sp. were tested the dispersion was mainly limited to Pectobacterium carotovorum biofilms, decreasing up to 15 % after exposure to molsidomine. Finally, the association of selected nitric oxide donors with sanitizers (DiQuat, H2O2, peracetic acid and PhenoTek II) was effective in dispersing biofilms. The best dispersal was achieved by pre-treating P. carotovorum with molsidomine and then peracetic acid. The synergistic effect was estimated up to ~35 % in dispersal when compared with peracetic acid alone. The association of nitric oxide donors with sanitizers could provide a foundation for an improved sanitization procedure for cleaning refrigerate environments

Absence of Staphylococcus aureus in Wild Populations of Fish Supports a Spillover Hypothesis

Staphylococcus aureus is a human commensal and opportunistic pathogen that also infects other animals. In humans and livestock, where S. aureus is most studied, strains are specialized for different host species. Recent studies have also found S. aureus in diverse wild animals. However, it remains unclear whether these isolates are also specialized for their hosts or whether their presence is due to repeated spillovers from source populations. This study focuses on S. aureus in fish, testing the spillover hypothesis in two ways. First, we examined 12 S. aureus isolates obtained from the internal and external organs of a farmed fish. While all isolates were from clonal complex 45, genomic diversity indicates repeated acquisition. The presence of a φSa3 prophage containing human immune evasion genes suggests that the source was originally human. Second, we tested for S. aureus in wild fish that were isolated from likely sources. In particular, we sampled 123 brown trout and their environment at 16 sites in the remote Scottish Highlands with variable levels of exposure to humans, birds, and livestock. This screen found no S. aureus infection in any of the wild populations or their environment. Together, these results support that the presence of S. aureus in fish and aquaculture is due to spillover from humans rather than specialization. Given the trends of increasing fish consumption, a better understanding of the dynamics of S. aureus spillover in aquaculture will mitigate future risks to fish and human health. IMPORTANCE Staphylococcus aureus is a human and livestock commensal but also an important pathogen responsible for high human mortality rates and economic losses in farming. Recent studies show that S. aureus is common in wild animals, including fish. However, we do not know whether these animals are part of the normal host range of S. aureus or whether infection is due to repeated spillover events from true S. aureus hosts. Answering this question has implications for public health and conservation. We find support for the spillover hypothesis by combining genome sequencing of S. aureus isolates from farmed fish and screens for S. aureus in isolated wild populations. The results imply that fish are unlikely to be a source of novel emergent S. aureus strains but highlight the prominence of the spillover of antibiotic-resistant bacteria from humans and livestock. This may affect both future fish disease potential and the risk of human food poisoning

Atypical cellular elements of unknown origin in the subbasal nerve plexus of a diabetic cornea diagnosed by large-area confocal laser scanning microscopy

In vivo large-area confocal laser scanning microscopy (CLSM) of the human eye using EyeGuidance technology allows a large-scale morphometric assessment of the corneal subbasal nerve plexus (SNP). Here, the SNP of a patient suffering from diabetes and associated late complications was analyzed. The SNP contained multiple clusters of large hyperintense, stellate-shaped, cellular-like structures. Comparable structures were not observed in control corneas from healthy volunteers. Two hypotheses regarding the origin of these atypical structures are proposed. First, these structures might be keratocyte-derived myofibroblasts that entered the epithelium from the underlying stroma through breaks in Bowman’s layer. Second, these structures could be proliferating Schwann cells that entered the epithelium in association with subbasal nerves. The nature and pathophysiological significance of these atypical cellular structures, and whether they are a direct consequence of the patient’s diabetic neuropathy/or a non-specific secondary effect of associated inflammatory processes, are unknown

Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen.

Funder: Raymond and Beverly Sackler FoundationFunder: Isaac Newton TrustFunder: Newnham College, University of CambridgeFunder: Medical Research CouncilMutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study, we investigate two factors that are predicted to influence the mutation rate: ecology and genome size. We conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact that ecology can have on the adaptive potential of bacterial pathogens

37. Multicenter, randomized study assessing the impact of amifostine on normal tissue radiation tolerance during head and neck cancer radiotherapy

A prospective, randomized multicenter study was conducted to assess the value of amifostine (Ethyol®) as a radioprotectant in head and neck cancer radiotherapy. The aim of the study was to evaluate the impact of the addition of daily amifostine (150 mg/m2) on the degree of early (mucositis, dysphagia, xerostomia) and late (mucosal, cutaneous, salivary gland, mandible and spinal cord) radiation reactions. Assessed were also patients’ quality of life, local control and overall survival. Sixty two patients from five Polish institutions were randomly assigned to radiotherapy alone (Arm A – 28 patients) or radiotherapy + amifostine (Arm B – 34 patients). There were 43 men and 19 women. Primary tumor was located in the oral cavity (27 patients), oropharynx (25 patients), nasopharynx (2 patients) and larynx/hypopharynx (8 patients). In 43 patients radiotherapy was used as the sole modality of treatment and 19 patients were irradiated postoperatively. The side effects of amifostine were manageable. In 6 patients amifostine infusion had to be temporarily stopped due to hypotension and in 5 patients its administration was permanently terminated due to hypotension, nausea and vomiting, septicemia or fever and visual disturbances. The early results of the study, focusing on early radiation reactions, will be presented at the conference

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.This work was primarily funded by an EU Horizon 2020 grant “PIGSs” (727966) and a ZELS BBSRC award “Myanmar Pigs Partnership (MPP)” (BB/L018934/1). G.G.R.M., E.L.M., and L.A.W. were supported by a Sir Henry Dale Fellowship to L.A.W. jointly funded by the Wellcome Trust and the Royal Society (109385/Z/15/Z). N.H. was supported by a Challenge grant from the Royal Society (CH16011) and an Isaac Newton Trust Research Grant [17.24(u)]. G.G.R.M. was also supported by a Research Fellowship at Newnham College. S.B. is supported by the Medical Research Council (MR/V032836/1). PIC North America provided part of the funds for the sequencing of the isolates from the USA. A.J.B. and M.M. were funded by Medical Research Council and Biotechnology and Biological Sciences Research Council studentships respectively, and M.M. was co-funded by the Raymond and Beverly Sackler Fund. We would like to acknowledge Susanna Williamson at the APHA for providing samples, Oscar Cabezón for sampling of the wild boar population in Spain, Mark O’Dea for access to sequence data from Australian isolates, the PIGSs and MPP consortiums for providing samples and helpful discussions, Julian Parkhill and John Welch for helpful discussions, and two anonymous reviewers for their valuable suggestions for improving the manuscript. This research was funded in whole or in part by the Wellcome Trust. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript (AAM) version arising from this submission.info:eu-repo/semantics/publishedVersio

Heat Resistance Mediated by a New Plasmid Encoded Clp ATPase, ClpK, as a Possible Novel Mechanism for Nosocomial Persistence of Klebsiella pneumoniae

Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably thermotolerant providing a conceivable explanation of its persistence in the hospital environment. This marked phenotype is mediated by a novel type of Clp ATPase, designated ClpK. The clpK gene is encoded by a conjugative plasmid and we find that the clpK gene alone renders an otherwise sensitive E. coli strain resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance of Clp ATPases in acquired environmental fitness and highlights the challenges of mobile genetic elements in fighting nosocomial infections

The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized