Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence.

Clostridiodes difficile (C. difficile) was ranked an "urgent threat" by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the "hypervirulent" strain, R20291, make it difficult to assign a "one size fits all" mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

Competent but complex communication: The phenomena of pheromone-responsive plasmids.

Enterococci are robust gram-positive bacteria that are found in a variety of surroundings and that cause a significant number of healthcare-associated infections. The genus possesses a high-efficiency pheromone-responsive plasmid (PRP) transfer system for genetic exchange that allows antimicrobial-resistance determinants to spread within bacterial populations. The pCF10 plasmid system is the best characterised, and although other PRP systems are structurally similar, they lack exact functional homologues of pCF10-encoded genes. In this review, we provide an overview of the enterococcal PRP systems, incorporating functional details for the less-well-defined systems. We catalogue the virulence-associated elements of the PRPs that have been identified to date, and we argue that this reinforces the requirement for elucidation of the less studied systems.

Enterococcal biofilm-A nidus for antibiotic resistance transfer?

Enterococci, which are on the WHO list of priority pathogens, are commonly encountered in hospital acquired infection and are becoming increasing significant due to the development of strains resistant to multiple antibiotics. Enterococci are also important microorganisms in the environment, and their presence is frequently used as an indicator of faecal pollution. Their success is related to their ability to survive within a broad range of habitats and the ease by which they acquire mobile genetic elements, including plasmids, from other bacteria. The enterococci are frequently present within a bacterial biofilm, which provides stability and protection to the bacterial population along with an opportunity for a variety of bacterial interactions. Enterococci can accept extrachromosomal DNA both from within its own species and from other bacterial species, and this is enhanced by the proximity of the donor and recipient strains. It is this exchange of genetic material that makes the role of biofilms such an important aspect of the success of enterococci. There remain many questions regarding the most suitable model systems to study enterococci in biofilms and regarding the transfer of genetic material including antibiotic resistance in these biofilms. This review focuses on some important aspects of biofilm in the context of horizontal gene transfer (HGT) in enterococci.

ATP7B variant penetrance explains differences between genetic and clinical prevalence estimates for Wilson disease.

Wilson disease (WD) is a genetic disorder of copper metabolism caused by variants in the copper transporting P-type ATPase gene ATP7B. Estimates for WD population prevalence vary with 1 in 30,000 generally quoted. However, some genetic studies have reported much higher prevalence rates. The aim of this study was to estimate the population prevalence of WD and the pathogenicity/penetrance of WD variants by determining the frequency of ATP7B variants in a genomic sequence database. A catalogue of WD-associated ATP7B variants was constructed, and then, frequency information for these was extracted from the gnomAD data set. Pathogenicity of variants was assessed by (a) comparing gnomAD allele frequencies against the number of reports for variants in the WD literature and (b) using variant effect prediction algorithms. 231 WD-associated ATP7B variants were identified in the gnomAD data set, giving an initial estimated population prevalence of around 1 in 2400. After exclusion of WD-associated ATP7B variants with predicted low penetrance, the revised estimate showed a prevalence of around 1 in 20,000, with higher rates in the Asian and Ashkenazi Jewish populations. Reanalysis of other recent genetic studies using our penetrance criteria also predicted lower population prevalences for WD in the UK and France than had been reported. Our results suggest that differences in variant penetrance can explain the discrepancy between reported epidemiological and genetic prevalences of WD. They also highlight the challenge in defining penetrance when assigning causality to some ATP7B variants.

Natural quorum sensing inhibitors effectively downregulate gene expression of Pseudomonas aeruginosa virulence factors.

At present, anti-virulence drugs are being considered as potential therapeutic alternatives and/or adjuvants to currently failing antibiotics. These drugs do not kill bacteria but inhibit virulence factors essential for establishing infection and pathogenesis through targeting non-essential metabolic pathways reducing the selective pressure to develop resistance. We investigated the effect of naturally isolated plant compounds on the repression of the quorum sensing (QS) system which is linked to virulence/pathogenicity in Pseudomonas aeruginosa. Our results show that trans-cinnamaldehyde (CA) and salicylic acid (SA) significantly inhibit expression of QS regulatory and virulence genes in P. aeruginosa PAO1 at sub-inhibitory levels without any bactericidal effect. CA effectively downregulated both the las and rhl QS systems with lasI and lasR levels inhibited by 13- and 7-fold respectively compared to 3- and 2-fold reductions with SA treatment, during the stationary growth phase. The QS inhibitors (QSI) also reduced the production of extracellular virulence factors with CA reducing protease, elastase and pyocyanin by 65%, 22% and 32%, respectively. The QSIs significantly reduced biofilm formation and concomitantly with repressed rhamnolipid gene expression, only trace amount of extracellular rhamnolipids were detected. The QSIs did not completely inhibit virulence factor expression and production but their administration significantly lowered the virulence phenotypes at both the transcriptional and extracellular levels. This study shows the significant inhibitory effect of natural plant-derived compounds on the repression of QS systems in P. aeruginosa.

A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides.

BACKGROUND: The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target. RESULTS: Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM. CONCLUSIONS: This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.

Biocontrol of Burkholderia cepacia complex bacteria and bacterial phytopathogens by Bdellovibrio bacteriovorus.

Bdellovibrio and like organisms are predatory bacteria that have the unusual property of using the cytoplasmic constituents of other Gram-negative bacteria as nutrients. These predators may thus provide an alternative approach to the biocontrol of human and plant pathogens. Predators were isolated on Burkholderia cenocepacia K56-2 and J2315 as prey cells, in enrichment cultures with soil and sewage. Three isolates (DM7C, DM8A, and DM11A) were identified as Bdellovibrio bacteriovorus on the basis of morphology, a periplasmic life cycle, and 16S rRNA gene sequencing. The prey range of these isolates was tested on Burkholderia cepacia complex bacteria and several phytopathogenic bacteria of agricultural importance. Of 31 strains of the Burkholderia cepacia complex tested, only 4 were resistant to predation by strain DM7C. A subset of 9 of the prey tested were also susceptible to strains DM8A and DM11A. Of 12 phytopathogens tested, 4 were resistant to strains DM7C and DM8A, and only 2 were resistant to strain DM11A. Thus, Bdellovibrio bacteriovorus strains retrieved from environmental samples on 2 Burkholderia cenocepacia isolates from cystic fibrosis patients did not distinguish in their prey range between other isolates of that pathogen or phytopathogens. Such strains hold promise as potential wide-spectrum biocontrol agents.

The ancient Britons: groundwater fauna survived extreme climate change over tens of millions of years across NW Europe.

Global climate changes during the Cenozoic (65.5-0 Ma) caused major biological range shifts and extinctions. In northern Europe, for example, a pattern of few endemics and the dominance of wide-ranging species is thought to have been determined by the Pleistocene (2.59-0.01 Ma) glaciations. This study, in contrast, reveals an ancient subsurface fauna endemic to Britain and Ireland. Using a Bayesian phylogenetic approach, we found that two species of stygobitic invertebrates (genus Niphargus) have not only survived the entire Pleistocene in refugia but have persisted for at least 19.5 million years. Other Niphargus species form distinct cryptic taxa that diverged from their nearest continental relative between 5.6 and 1.0 Ma. The study also reveals an unusual biogeographical pattern in the Niphargus genus. It originated in north-west Europe approximately 87 Ma and underwent a gradual range expansion. Phylogenetic diversity and species age are highest in north-west Europe, suggesting resilience to extreme climate change and strongly contrasting the patterns seen in surface fauna. However, species diversity is highest in south-east Europe, indicating that once the genus spread to these areas (approximately 25 Ma), geomorphological and climatic conditions enabled much higher diversification. Our study highlights that groundwater ecosystems provide an important contribution to biodiversity and offers insight into the interactions between biological and climatic processes.

How suitable is freshwater sponge Ephydatia fluviatilis (Linnaeus, 1759) for time-integrated biomonitoring of microbial water quality?

Faecal pollution of water by bacteria has a negative effect on water quality and can pose a potential health hazard. Conventional surveillance of microbial water quality relies on the analysis of low-frequency spot samples and is thus likely to miss episodic or periodic pollution. This study aimed to investigate the potential of filter-feeding sponges for time-integrated biomonitoring of microbial water quality. Laboratory trials tested the effects of different ratios of bacterial abundance and the sequence of exposure on bacterial retention by the freshwater sponge Ephydatia fluviatilis (Linnaeus, 1759) to establish its potential to indicate bacterial exposure. Gemmule grown sponges were simultaneously exposed to Escherichia coli and Enterococcus faecalis but at different ratios (Trial 1) or individually exposed to each bacterial species but in different sequential order (Trial 2). The E. coli and E. faecalis retained in each sponge was quantified by culture on selective agars. Data analysis was conducted using the Kruskal-Wallis test and/or the Mann-Whitney U test to compare between the numbers of bacteria retained in each treatment. Additionally, the Wilcoxon matched-paired signed-rank test was used for comparison of the different bacterial abundances retained within each individual sponge. Sponges from all trials retained E. coli and E. faecalis in small numbers relative to the exposure (<0.05 % Trial 1 and <0.07 % Trial 2) but exhibited higher retention of E. coli. Higher abundance of either bacterial species resulted in significantly lower (P<0.005) retention of the same species within sponges (Trial 1). An initial exposure to E. coli resulted in significantly higher (P=0.040) retention of both bacterial species than when sponges were exposed to E. faecalis first (Trial 2).Bacterial retention by sponges was neither quantitatively representative of bacterial abundance in the ambient water nor the sequence of exposure. This implies either selective filtration or an attempt by sponges to prevent infection. However, freshwater sponges may still be useful in biomonitoring as qualitative time-integrated samplers of faecal indicator bacteria as they detect different bacteria present in the water even if their quantities cannot be estimated.

Microbial Biosurfactants: Antimicrobial Activity and Potential Biomedical and Therapeutic Exploits.

The rapid emergence of multidrug-resistant pathogens worldwide has raised concerns regarding the effectiveness of conventional antibiotics. This can be observed in ESKAPE pathogens, among others, whose multiple resistance mechanisms have led to a reduction in effective treatment options. Innovative strategies aimed at mitigating the incidence of antibiotic-resistant pathogens encompass the potential use of biosurfactants. These surface-active agents comprise a group of unique amphiphilic molecules of microbial origin that are capable of interacting with the lipidic components of microorganisms. Biosurfactant interactions with different surfaces can affect their hydrophobic properties and as a result, their ability to alter microorganisms' adhesion abilities and consequent biofilm formation. Unlike synthetic surfactants, biosurfactants present low toxicity and high biodegradability and remain stable under temperature and pH extremes, making them potentially suitable for targeted use in medical and pharmaceutical applications. This review discusses the development of biosurfactants in biomedical and therapeutic uses as antimicrobial and antibiofilm agents, in addition to considering the potential synergistic effect of biosurfactants in combination with antibiotics. Furthermore, the anti-cancer and anti-viral potential of biosurfactants in relation to COVID-19 is also discussed.

Biological and synthetic surfactant exposure increases antimicrobial gene occurrence in a freshwater mixed microbial biofilm environment.

Aquatic habitats are particularly susceptible to chemical pollution, such as antimicrobials, from domestic, agricultural, and industrial sources. This has led to the rapid increase of antimicrobial resistance (AMR) gene prevalence. Alternate approaches to counteract pathogenic bacteria are in development including synthetic and biological surfactants such as sodium dodecyl sulfate (SDS) and rhamnolipids. In the aquatic environment, these surfactants may be present as pollutants with the potential to affect biofilm formation and AMR gene occurrence. We tested the effects of rhamnolipid and SDS on aquatic biofilms in a freshwater stream in Northern Ireland. We grew biofilms on contaminant exposure substrates deployed within the stream over 4 weeks. We then extracted DNA and carried out shotgun sequencing using a MinION portable sequencer to determine microbial community composition, with 16S rRNA analyses (64,678 classifiable reads identified), and AMR gene occurrence (81 instances of AMR genes over 9 AMR gene classes) through a metagenomic analysis. There were no significant changes in community composition within all systems; however, biofilm exposed to rhamnolipid had a greater number of unique taxa as compared to SDS treatments and controls. AMR gene prevalence was higher in surfactant-treated biofilms, although not significant, with biofilm exposed to rhamnolipids having the highest presence of AMR genes and classes compared to the control or SDS treatments. Our results suggest that the presence of rhamnolipid encourages an increase in the prevalence of AMR genes in biofilms produced in mixed-use water bodies.

Whole genome RNA expression profiling of endoscopic biliary brushings provides data suitable for biomarker discovery in cholangiocarcinoma.

BACKGROUND & AIMS: Molecular analyses of biliary brushings using microarray and qPCR have the potential to provide valuable information on the biology of biliary diseases. Microarray analysis of biliary strictures has rarely been applied to endoscopic biliary brushings. METHODS: Biliary brushings were obtained from patients with benign and malignant biliary disease at the time of ERCP. Microarray analysis of mRNA isolated using brushings from 10 patients was validated for a selection of genes by qPCR using the same source mRNA and a second fresh set of nine biliary brushings as well as surgical resection tissue. Cultured cholangiocytes were used to assess the impact of bile or X-ray contrast solution on RNA quality. RESULTS: RNA was of variable quantity (100-1500 ng) and poor quality (Agilent RNA Integrity Number (RIN)<5, estimated to be fragments 100 to 600 base pairs long). Reliable qPCR results required primer pairs designed to produce amplicons <130 bp. Differential gene expression by microarray analysis identified 1140 up-regulated genes and 1001 down-regulated genes between benign and malignant biliary strictures. The trends in a selection of 45 up-regulated genes, including various HOX genes, collagens, PVT1, MUC4, MUC5AC, and LEF1, were validated by qPCR using RNA from biliary strictures with a moderate to strong correlation coefficient between microarray and qPCR (r=0.41 to r=0.57). Immunohistochemistry of surgical resection tissues (n=23) showed elevated CD9, SERPINA3, and PNMA2 protein expression in cancer samples. CONCLUSIONS: RNA isolated from biliary brushings is suitable for molecular analysis of biliary diseases using qPCR and microarray.

UVc-irradiation sublethal stress does not alter antibiotic susceptibility of staphylococci (MRSA, MSSA, and coagulase-negative staphylococci) to ß-lactam, macrolide, and fluoroquinolone antibiotic agents.

Skin tanning, either by exposure to natural sunlight or through use of UV sunbeds, has become a popular practice in the US, where it is estimated that approximately 1 million times per day someone in the US uses UV radiation for skin tanning, equating to 30 million Americans (circa 10% of the US population) who use a tanning bed. As well as exposing the host to periods of UV radiation, such practices also expose commensal skin bacteria, including Staphylococcus aureus, to such UV radiation. Previous work has indicated that environmental stresses on bacteria may lead to an upregulation of stress responses, in an attempt for the organism to combat the applied stress and remain viable. UV light may act as an environmental stress on bacteria, and so it was the aim of this study to examine the effect of UVc light on the antibiotic susceptibility of commensal skin bacteria, to determine if UV radiation would increase the antibiotic resistance of such skin flora and thus lead to a potential skin flora with increased antibiotic resistance. Previously, it has been shown that UVc light has a greater mutational effect on bacteria compared to lower-energy UV forms, including UVa and UVb light. Therefore, we decided to employ UVc light in our study to amplify the potential for mutational events occurring in skin staphylococci organisms (n=8) including methicillin-sensitive Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (n=4), and coagulase-negative staphylococci (Staphylococcus haemolyticus) (n=2) were exposed to varying degrees of sublethal radiation via UVc light, and their minimum inhibitory concentration (MIC) susceptibility was determined by broth dilution assay against three classes of commonly used antibiotics, namely ß-lactams (penicillin), macrolides (erythromycin), and fluoroquinolones (ciprofloxacin). There was no significant difference between antibiotic susceptibility before UVc exposure and until maximum sublethal stress, prior to cell death due to fatal UVc exposure with the cells. These results indicate that UV environmental stress/exposure does not upregulate antibiotic resistance, and therefore these data indicate that UVc radiation does not lead to a more antibiotic-resistant population in the staphylococci organisms post-exposure.

Molecular characterization and phylogenetic analysis of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE gene loci in viridans group streptococci isolated from adult patients with cystic fibrosis.

OBJECTIVES: Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs). METHODS: The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis. RESULTS: Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ≥8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%). CONCLUSIONS: Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.

Nasopharyngeal Swabs vs. Nasal Aspirates for Respiratory Virus Detection: A Systematic Review.

Nasal pathogen detection sensitivities can be as low as 70% despite advances in molecular diagnostics. This may be linked to the choice of sampling method. A diagnostic test accuracy review for sensitivity was undertaken to compare sensitivity of swabbing to the nasopharynx and extracting nasal aspirates, using the PRISMA protocol, Cochrane rapid review methodology, and QUADAS-2 risk of bias tools, with meta-analysis of included studies. Sensitivities were calculated by a consensus standard of positivity by either method as the 'gold standard.' Insufficient sampling methodology, cross sectional study designs, and studies pooling samples across anatomical sites were excluded. Of 13 subsequently eligible studies, 8 had 'high' risk of bias, and 5 had 'high' applicability concerns. There were no statistical differences in overall sensitivities between collection methods for eight different viruses, and this did not differ with use of PCR, immunofluorescence, or culture. In one study alone, Influenza H1N1(2009) favored nasopharyngeal swabs, with aspirates having 93.3% of the sensitivity of swabs (p > 0.001). Similarly equivocal sensitivities were noted in reports detecting bacteria. The chain of sampling, from anatomical site to laboratory results, features different potential foci along which sensitivity may be lost. A fair body of evidence exists that use of a different sampling method will not yield more respiratory pathogens.

A Novel Biofilm Model System to Visualise Conjugal Transfer of Vancomycin Resistance by Environmental Enterococci.

Enterococci and biofilm-associated infections are a growing problem worldwide, given the rise in antibiotic resistance in environmental and clinical settings. The increasing incidence of antibiotic resistance and its propagation potential within enterococcal biofilm is a concern. This requires a deeper understanding of how enterococcal biofilm develops, and how antibiotic resistance transfer takes place in these biofilms. Enterococcal biofilm assays, incorporating the study of antibiotic resistance transfer, require a system which can accommodate non-destructive, real-time experimentation. We adapted a Gene Frame® combined with fluorescence microscopy as a novel non-destructive platform to study the conjugal transfer of vancomycin resistance in an established enterococcal biofilm.A multi-purpose fluorescent in situ hybridisation (FISH) probe, in a novel application, allowed the identification of low copy number mobile elements in the biofilm. Furthermore, a Hoechst stain and ENU 1470 FISH probe identified Enterococcus faecium transconjugants by excluding Enterococcus faecalis MF06036 donors. Biofilm created with a rifampicin resistant E. faecalis (MW01105Rif) recipient had a transfer efficiency of 2.01 × 10-3; double that of the biofilm primarily created by the donor (E. faecalis MF06036). Conjugation in the mixed enterococcal biofilm was triple the efficiency of donor biofilm. Double antibiotic treatment plus lysozyme combined with live/dead imaging provided fluorescent micrographs identifying de novo enterococcal vancomycin resistant transconjugants inside the biofilm. This is a model system for the further study of antibiotic resistance transfer events in enterococci. Biofilms promote the survival of enterococci and reduce the effectiveness of drug treatment in clinical settings, hence giving enterococci an advantage. Enterococci growing in biofilms exchange traits by means of horizontal gene transfer, but currently available models make study difficult. This work goes some way to providing a non-destructive, molecular imaging-based model system for the detection of antibiotic resistance gene transfer in enterococci.

The Fate of Foodborne Pathogens in Manure Treated Soil.

The aim of this review was to provide an update on the complex relationship between manure application, altered pathogen levels and antibiotic resistance. This is necessary to protect health and improve the sustainability of this major farming practice in agricultural systems based on high levels of manure production. It is important to consider soil health in relation to environment and land management practices in the context of the soil microflora and the introduction of pathogens on the health of the soil microbiome. Viable pathogens in manure spread on agricultural land may be distributed by leaching, surface run-off, water source contamination and contaminated crop removal. Thus it is important to understand how multiple pathogens can persist in manures and on soil at farm-scale and how crops produced under these conditions could be a potential transfer route for zoonotic pathogens. The management of pathogen load within livestock manure is a potential mechanism for the reduction and prevention of outbreaks infection with Escherichia coli, Listeria Salmonella, and Campylobacter. The ability of Campylobacter, E. coli, Listeria and Salmonella to combat environmental stress coupled with their survival on food crops and vegetables post-harvest emphasizes the need for further study of these pathogens along with the emerging pathogen Providencia given its link to disease in the immunocompromised and its' high levels of antibiotic resistance. The management of pathogen load within livestock manure has been widely recognized as a potential mechanism for the reduction and prevention of outbreaks infection but any studies undertaken should be considered as region specific due to the variable nature of the factors influencing pathogen content and survival in manures and soil. Mediocre soils that require nutrients could be one template for research on manure inputs and their influence on soil health and on pathogen survival on grassland and in food crops.

Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing.

Pseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI's) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

Low-Molecular-Weight Seaweed-Derived Polysaccharides Lead to Increased Faecal Bulk but Do Not Alter Human Gut Health Markers.

Seaweeds are potentially sustainable crops and are receiving significant interest because of their rich bioactive compound content; including fatty acids, polyphenols, carotenoids, and complex polysaccharides. However, there is little information on the in vivo effects on gut health of the polysaccharides and their low-molecular-weight derivatives. Herein, we describe the first investigation into the prebiotic potential of low-molecular-weight polysaccharides (LMWPs) derived from alginate and agar in order to validate their in vivo efficacy. We conducted a randomized; placebo-controlled trial testing the impact of alginate and agar LWMPs on faecal weight and other markers of gut health and on composition of gut microbiota. We show that these LMWPs led to significantly increased faecal bulk (20-30%). Analysis of gut microbiome composition by sequencing indicated no significant changes attributable to treatment at the phylum and family level, although FISH analysis showed an increase in Faecalibacterium prausnitzii in subjects consuming agar LMWP. Sequence analysis of gut bacteria corroborated with the FISH data, indicating that alginate and agar LWMPs do not alter human gut microbiome health markers. Crucially, our findings suggest an urgent need for robust and rigorous human in vivo testing-in particular, using refined seaweed extracts.

Pseudomonas aeruginosa cystic fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro.

BACKGROUND: Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro. RESULTS: Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture. CONCLUSIONS: Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows "non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung.