Search

Salmonella enterica and Escherichia coli use the inner membrane transporter DctA to import the pyrimidine biosynthetic pathway intermediate orotate from the environment. To study the regulation of dctA expression, we used an S. enterica serovar Typhimurium pyrimidine auxotroph to select a mutant that could grow in an otherwise nonpermissive culture medium containing glucose and a low concentration of orotate. Whole genome sequencing revealed a point mutation upstream of dctA in the putative cyclic AMP receptor protein (CRP) binding site. The C→T transition converted the least favourable base to the most favourable base for CRP–DNA affinity. A dctA::lux transcriptional fusion confirmed that the mutant dctA promoter gained responsiveness to CRP even in the presence of glucose. Moreover, dctA expression was higher in the mutant than the wild type in the presence of alternative carbon sources that activate CRP.

The bacterium Coxiella burnetii can cause the disease Q-fever in a wide range of animal hosts. Ruminants, including sheep, are thought to play a pivotal role in the transmission of C. burnetii to humans; however, the only existing livestock vaccine, namely, Coxevac® (Ceva Animal Health Ltd., Libourne, France), a killed bacterin vaccine based on phase I C. burnetii strain Nine-Mile, is only approved for use in goats and cattle. In this study, a pregnant ewe challenge model was used to determine the protective effects of Coxevac® and an experimental bacterin vaccine based on phase II C. burnetii against C. burnetii challenge. Prior to mating, ewes (n = 20 per group) were vaccinated subcutaneously with either Coxevac®, the phase II vaccine, or were unvaccinated. A subset of pregnant ewes (n = 6) from each group was then challenged 151 days later (~100 days of gestation) with 106 infectious mouse doses of C. burnetii, Nine-Mile strain RSA493. Both vaccines provided protection against C. burnetii challenge as measured by reductions in bacterial shedding in faeces, milk and vaginal mucus, and reduced abnormal pregnancies, compared to unvaccinated controls. This work highlights that the phase I vaccine Coxevac® can protect ewes against C. burnetii infection. Furthermore, the phase II vaccine provided comparable levels of protection and may offer a safer and cost-effective alternative to the currently licensed vaccine.

H-NS is a nucleoid structuring protein and global repressor of virulence and horizontally-acquired genes in bacteria. H-NS can interact with itself or with homologous proteins, but protein family diversity and regulatory network overlap remain poorly defined. Here, we present a comprehensive phylogenetic analysis that revealed deep-branching clades, dispelling the presumption that H-NS is the progenitor of varied molecular backups. Each clade is composed exclusively of either chromosome-encoded or plasmid-encoded proteins. On chromosomes, stpA and newly discovered hlpP are core genes in specific genera, whereas hfp and newly discovered hlpC are sporadically distributed. Six clades of H-NS plasmid proteins (Hpp) exhibit ancient and dedicated associations with plasmids, including three clades with fidelity for plasmid incompatibility groups H, F or X. A proliferation of H-NS homologs in Erwiniaceae includes the first observation of potentially co-dependent H-NS forms. Conversely, the observed diversification of oligomerization domains may facilitate stable co-existence of divergent homologs in a genome. Transcriptomic and proteomic analysis in Salmonella revealed regulatory crosstalk and hierarchical control of H-NS homologs. We also discovered that H-NS is both a repressor and activator of Salmonella Pathogenicity Island 1 gene expression, and both regulatory modes are restored by Sfh (HppH) in the absence of H-NS.

Wastewater based epidemiology (WBE) has become an important tool during the COVID-19 pandemic, however the relationship between SARS-CoV-2 RNA in wastewater treatment plant influent (WWTP) and cases in the community is not well-defined. We report here the development of a national WBE program across 28 WWTPs serving 50% of the population of Scotland, including large conurbations, as well as low-density rural and remote island communities. For each WWTP catchment area, we quantified spatial and temporal relationships between SARS-CoV-2 RNA in wastewater and COVID-19 cases. Daily WWTP SARS-CoV-2 influent viral RNA load, calculated using daily influent flow rates, had the strongest correlation (ρ > 0.9) with COVID-19 cases within a catchment. As the incidence of COVID-19 cases within a community increased, a linear relationship emerged between cases and influent viral RNA load. There were significant differences between WWTPs in their capacity to predict case numbers based on influent viral RNA load, with the limit of detection ranging from 25 cases for larger plants to a single case in smaller plants. SARS-CoV-2 viral RNA load can be used to predict the number of cases detected in the WWTP catchment area, with a clear statistically significant relationship observed above site-specific case thresholds., There is a strong, site specific, relationship between COVID-19 cases and SARS-CoV-2 viral RNA load in wastewater treatment plant influent.

The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.

SummaryBackgroundCOVID-19 patients shed SARS-CoV-2 RNA in their faeces. We hypothesised that detection of SARS-CoV-2 RNA in wastewater treatment plant (WWTP) influent could be a valuable tool to assist in public health decision making. We aimed to rapidly develop and validate a scalable methodology for the detection of SARS-CoV-2 RNA in wastewater that could be implemented at a national level and to determine the relationship between the wastewater signal and COVID-19 cases in the community.MethodsWe developed a filtration-based methodology for the concentration of SARS-CoV-2 from WWTP influent and subsequent detection and quantification by RT-qPCR. This methodology was used to monitor 28 WWTPs across Scotland, serving 50% of the population. For each WWTP catchment area, we collected data describing COVID-19 cases and deaths. We quantified spatial and temporal relationships between SARS-CoV-2 RNA in wastewater and COVID-19 cases.FindingsDaily WWTP SARS-CoV-2 influent viral RNA load, calculated using daily influent flow rates, had the strongest correlation (ρ>0.9) with COVID-19 cases within a catchment. As the incidence of COVID-19 cases within a community increased, a linear relationship emerged between cases and influent viral RNA load. There were significant differences between WWTPs in their capacity to predict case numbers based on influent viral RNA load, with the limit of detection ranging from twenty-five cases for larger plants to a single case in smaller plants.InterpretationThe levels of SARS-CoV-2 RNA in WWTP influent provide a cost-effective and unbiased measure of COVID-19 incidence within a community, indicating that national scale wastewater-based epidemiology can play a role in COVID-19 surveillance. In Scotland, wastewater testing has been expanded to cover 75% of the population, with sub-catchment sampling being used to focus surge testing. SARS-CoV-2 variant detection, assessment of vaccination on community transmission and surveillance for other infectious diseases represent promising future applications.FundingThis study was funded by project grants from the Scottish Government via the Centre of Expertise for Waters (CD2019/06) and The Natural Environment Research Council’s COVID-19 Rapid Response grants (NE/V010441/1). The Roslin Institute receives strategic funding from the Biotechnology and Biological Sciences Research Council (BB/P013740/1, BBS/E/D/20002173). Sample collection and supplementary analysis was funded and undertaken by Scottish Water and the majority of the sample analysis was funded and undertaken by the Scottish Environment Protection Agency.

The human zoonotic pathogen Escherichia coli O157 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157 serogroup and defines the phenotypic consequences of specific structural variants. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157 and by using both optical mapping and ONT long-read sequencing demonstrate that LCRs are generated in laboratory cultures started from a single colony and particular variants are selected during animal colonisation. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga toxin production, type 3 secretion and motility are affected by LCRs. In summary, E. coli O157 has acquired multiple prophage regions over time that act as genome engineers to continually produce structural variants of the genome. This structural variation is a form of epigenetic regulation that generates sub-population phenotypic heterogeneity with important implications for bacterial adaptation and survival.Author SummaryEscherichia coli has an ‘open genome’ and has acquired genetic information over evolutionary time, often in the form of bacteriophages that integrate into the bacterial genome (prophages). E. coli O157 is a clonal serogroup that is found primarily in ruminants such as cattle but can cause life-threatening infections in humans. E. coli O157 isolates contain multiple prophages including those that encode Shiga-like toxins which are responsible for the more serious disease associated with human infections. We show in this study that many of these prophages exhibit large regions of sequence similarity that allow rearrangements to occur in the genome generating structural variants. These occur routinely during bacterial culture in the laboratory and the variants are detected during animal colonization. The variants generated can give the bacteria altered phenotypes, such as increased motility or toxin production which can be selected in specific environments and therefore represent a highly dynamic mechanism to generate variation in bacterial populations without a change in overall gene content.

Host adaptation of pathogens may increase intra- and interspecies transmission. We showed previously that the passage of a clinically isolated enterohemorrhagic Escherichia coli (EHEC) O157 strain (125/99) through the gastrointestinal tract of mice increases its pathogenicity in the same host. In this work, we aimed to elucidate the underlying mechanism(s) involved in the patho-adaptation of the stool-recovered (125RR) strain. We assessed the global transcription profile by microarray and found almost 100 differentially expressed genes in 125RR strain compared with 125/99 strain. We detected an overexpression of Type Three Secretion System (TTSS) proteins at the mRNA and protein levels and demonstrated increased adhesion to epithelial cell lines for the 125RR strain. Additional key attributes of the 125RR strain were: increased motility on semisolid agar, which correlated with an increased fliC mRNA level; reduced Stx2 production at the mRNA and protein levels; increased survival at pH 2.5, as determined by acid resistance assays. We tested whether the overexpression of the LEE-encoded regulator (ler) in trans in the 125/99 strain could recreate the increased pathogenicity observed in the 125RR strain. As anticipated ler overexpression led to increased expression of TTSS proteins and bacterial adhesion to epithelial cells in vitro but also increased mortality and intestinal colonization in vivo. We conclude that this host-adaptation process required changes in several mechanisms that improved EHEC O157 fitness in the new host. The research highlights some of the bacterial mechanisms required for horizontal transmission of these zoonotic pathogens between their animal and human populations.

To decouple the effects of collagen fiber density and network mechanics on cancer cell behavior, we describe a highly tunable in vitro 3D interpenetrating network (IPN) consisting of a primary fibrillar collagen network reinforced by a secondary visible light-mediated thiol-ene poly(ethylene glycol) (PEG) network. This PEG/Collagen IPN platform is cytocompatible, inherently bioactive via native cellular adhesion sites, and mechanically tunable over several orders of magnitude—mimicking both healthy and cancerous breast tissue. Furthermore, we use the PEG/Collagen IPN platform to investigate the effect of mechanical confinement on cancer cell behavior as it is hypothesized that cells within tumors that have yet to invade into the surrounding tissue experience mechanical confinement. We find that mechanical confinement via the IPN impairs behavior characteristic of malignant cells (i.e., viability, proliferation, and cellular motility) in the triple negative breast cancer cell line MDA.MB.231, and is more effective than removal of soluble growth signals. The PEG/Collagen IPN platform is a useful tool for studying mechanotransductive signaling pathways and motivates further investigation into the role of mechanical confinement in cancer progression. Statement of Significance In this study, we have developed, optimized, and applied a novel 3D in vitro cell culture platform composed of an interpenetrating network (IPN) that is both mechanically tunable and inherently bioactive. The IPN consists of a primary fibrillar collagen type-1 network reinforced by a secondary thiol-ene poly(ethylene glycol) (PEG) network. The IPNs are formed via a novel strategy in which cell-laden collagen gels are formed first, and soluble PEG monomers are added later and crosslinked via visible light. This approach ensures that the collagen gels contain a fibrillar architecture similar to the collagen architecture present in vivo. We applied our IPN platform to study the effect of mechanical confinement on cancer cell behavior and found that it inhibits malignant-like behavior.

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium., Author summary Enterohaemorrhagic E. coli (EHEC) O157 strains are found in cattle where they are asymptomatic, while human exposure can lead to severe symptoms including bloody diarrhoea and kidney damage due to the activity of Shiga toxin (Stx). The most serious symptoms in humans are associated with isolates that encode Stx subtype 2a. The advantage of these toxins in the animal reservoir is still not clear, however there is experimental evidence implicating Stx with increased bacterial adherence, immune modulation and suppression of predatory protozoa. In this study, the hypothesis that Stx2a is important for super-shedding and calf-to-calf transmission was tested by comparing excretion and transmission dynamics of E. coli O157 strains with and without Stx2a. While Stx2a did not alter excretion levels when calfs were orally challenge, it enabled colonisation of more in contact ‘sentinel’ animals in our transmission model. We show that Stx2a is generally induced more rapidly than Stx2c, resulting in increased levels of Stx2a expression. Both Stx2a and Stx2c were able to restrict cellular proliferation of epithelial cells in cultured bovine enteroids. Taken together, we propose that rapid production of Stx2a and its role in establishing E. coli O157 colonisation in the bovine gastrointestinal tract facilitate effective transmission and have led to its expansion in the cattle E. coli O157 population.

Despite advances in laboratory design, professional training, and workplace biosafety guidelines, laboratory-acquired infections continue to occur. Effective tools are required to investigate cases and prevent future illness. Here, we demonstrate the value of whole-genome sequencing as a tool for the identification and source attribution of laboratory-acquired salmonellosis.

The Sxy (TfoX) protein is required for expression of a distinct subset of the genes regulated by the cAMP receptor protein (CRP) in the model organisms Escherichia coli, Haemophilus influenzae, and Vibrio cholerae. Genetic studies have established that CRP and Sxy co-activate transcription at gene promoters containing DNA binding sites called CRP-S sites. In contrast, CRP acts without Sxy at gene promoters containing canonical CRP-N sites, suggesting that Sxy makes physical contacts with CRP and/or DNA to assist in transcriptional activation at CRP-S promoters. Despite growing interest in Sxy’s activity as a transcription factor, Sxy remains poorly characterized due to a lack of reliable phenotypes in E. coli. Experiments are further hampered by growth inhibition and formation of inclusion bodies when Sxy is overexpressed. In this study we applied diverse phenotypic and molecular assays to test for postulated Sxy functions and interactions. Mutations in conserved regions of Sxy and truncations in the Sxy C-terminus abolish transcriptional activation of a CRP-S promoter, and a 37 amino acid truncation of the C-terminus relieves the growth inhibition normally caused by Sxy overexpression. Sxy was unable to augment weakened CRP interactions to restore carbon metabolism phenotypes. Bandshift analysis and chromatin pull-down assays of Sxy-CRP-DNA interactions yielded intriguing evidence of CRP-Sxy and Sxy-DNA physical interactions. However, despite the careful application of standard protein purification protocols and quality control steps for nickel affinity column purification, protein mass spectrometry revealed the enrichment of additional DNA-binding proteins in nickel column eluates, presenting a probable source of artefactual protein-protein and protein-DNA interaction results. These findings highlight the importance of extensive controls and phenotypic assays for the study of poorly characterized and recalcitrant proteins like Sxy.

Objectives Ireland has the highest rate of vancomycin-resistant Enterococcus faecium (VREfm) isolated from blood of nosocomial patients in Europe, which rose from 33% (110/330) in 2007 to 45% (178/392) in 2012. No other European country had a VREfm rate from blood cultures of >25%. Our aim was to elucidate the reasons for this significantly higher rate in Ireland. Methods The epidemiology and molecular typing of VRE from bloodstream infections (BSIs) was examined in a tertiary care referral hospital and isolates were compared with those from other tertiary care referral centres in the region. Results The most common source of VRE BSIs was intra-abdominal sepsis, followed by line-related infection and febrile neutropenia. Most of the isolates were positive for vanA; 52% (43/83) possessed the esp gene and 12% (10/83) possessed the hyl gene. Genotyping by SmaI macrorestriction analysis (PFGE) of isolates revealed clonal relatedness between bloodstream isolates and environmental isolates. VRE BSI isolates from two other tertiary care hospitals in the Dublin region showed relatedness by PFGE analysis. MLST revealed four STs (ST17, ST18, ST78 and ST203), all belonging to the clonal complex of hospital-associated strains. Conclusions Irish VRE BSI isolates have virulence factor profiles as previously reported from Europe. Typing analysis shows the spread of individual clones within the hospital and between regional tertiary care hospitals. Apart from transmission of VRE within the hospital and transfer of colonized patients between Irish hospitals, no other explanation for the persistently high VREfm BSI rate in Ireland has been found.

The genome of Salmonella enterica subspecies enterica serovar Enteritidis phage type 8 strain EN1660, isolated from an outbreak in Thunder Bay, Canada, was sequenced to 46-fold coverage using an Illumina MiSeq with 300-bp paired-end sequencing chemistry to produce 28 contigs with an N 50 value of 490,721 bp.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a public health threat and outbreaks occur worldwide. Here, we investigate genomic differences between related STEC O157:H7 that caused two outbreaks, eight weeks apart, at the same restaurant. Short-read genome sequencing divided the outbreak strains into two sub-clusters separated by only three single-nucleotide polymorphisms in the core genome while traditional typing identified them as separate phage types, PT8 and PT54. Isolates did not cluster with local strains but with those associated with foreign travel to the Middle East/North Africa. Combined long-read sequencing approaches and optical mapping revealed that the two outbreak strains had undergone significant microevolution in the accessory genome with prophage gain, loss and recombination. In addition, the PT54 sub-type had acquired a 240 kbp multi-drug resistance (MDR) IncHI2 plasmid responsible for the phage type switch. A PT54 isolate had a general fitness advantage over a PT8 isolate in rich medium, including an increased capacity to use specific amino acids and dipeptides as a nitrogen source. The second outbreak was considerably larger and there were multiple secondary cases indicative of effective human-to-human transmission. We speculate that MDR plasmid acquisition and prophage changes have adapted the PT54 strain for human infection and transmission. Our study shows the added insights provided by combining whole-genome sequencing approaches for outbreak investigations.

Here, we report the first draft genome sequence of Salmonella enterica subsp. enterica serovar Livingstone. This S . Livingstone strain CKY-S4 displayed biofilm formation and cellulose production and could persist on lettuce. This genome may help the study of mechanisms by which enteric pathogens colonize food crops.

Knowledge of global regulatory networks has been exploited to rewire the gene control programmes of the model bacterium Salmonella enterica serovar Typhimurium. The product is an organism with competitive fitness that is superior to that of the wild type but tuneable under specific growth conditions. The paralogous hns and stpA global regulatory genes are located in distinct regions of the chromosome and control hundreds of target genes, many of which contribute to stress resistance. The locations of the hns and stpA open reading frames were exchanged reciprocally, each acquiring the transcription control signals of the other. The new strain had none of the compensatory mutations normally associated with alterations to hns expression in Salmonella; instead it displayed rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon. Thus the expression patterns of global regulators can be adjusted artificially to manipulate microbial physiology, creating a new and resilient organism.

An outbreak of linezolid-resistant vancomycin-resistant Enterococcus faecium (LRVREfm) occurred in the hepatology ward of a tertiary referral hospital in Ireland between February and September 2014. LRVREfm was isolated from 15 patients; pulsed-field gel electrophoresis confirmed spread of a single clone. This is the first report of an outbreak of linezolid-resistant vancomycin-resistant enterococcus in Ireland.

Summary Background Primary varicella infection is caused by varicella-zoster virus (VZV). It is a common childhood infection, which is usually benign but can occasionally cause morbidity and mortality. In immunosuppressed adults, atypical presentation and disseminated disease can occur with significant morbidity and mortality. A VZV vaccine is available. Objectives This study was designed to measure the prevalence of immunity to VZV and to determine the predictive value of a self-reported history of varicella infection in a population of dermatological patients receiving systemic immunosuppressant therapy. We sought to assess the need for routine serological testing for varicella-zoster immunity in this cohort. Methods Serological testing for VZV immunity was done on 228 patients receiving systemic immunosuppressive treatment for a dermatological condition. Information regarding a history of previous primary VZV infection was obtained from each patient. Results Two hundred and twenty-eight patients had VZV serology performed. The mean age of the patients was 49·6 years. The prevalence of VZV seropositivity in this cohort was 98·7%. One hundred and two patients (44·7%) reported having a definite history of primary VZV. The sensitivity of a self-reported history of VZV infection was 45·3% with a specificity of 100%. The positive and negative predictive values of a self-reported history of VZV for serologically confirmed immunity were 100% and 2·3%, respectively. Conclusions The prevalence of VZV IgG antibodies in our cohort of Irish dermatology patients receiving immunosuppressive therapy is 98·7%. A recalled history of varicella infection is a good predictor of serological immunity. This study has shown that there are VZV-susceptible individuals within our cohort. These patients did not have a clear history of previous infection. We recommend serological testing of patients without a clear history of infection prior to the commencement of immunosuppressive therapy and vaccination of patients with negative serology.

The process of IgE switching requires the prior transcription of the unrearranged Cepsilon gene, which leads to its recombination with the VDJ region. The activation of NF-kappaB by CD40 is a key process in facilitating this transcription by promoting the activation of the Cepsilon promoter. The present study explores the uncharacterized signalling pathways employed by CD40 in activating NF-kappaB by the overexpression of genes encoding wild-type and dominant-negative forms of the signalling components tumour-necrosis-factor-receptor-associated factor 6 (TRAF-6), NF-kappaB-inducing kinase (NIK), IkappaB kinase (IKK)-1 and IKK-2 in the BJAB B-cell line. The overexpression of TRAF-6 or NIK was sufficient to activate NF-kappaB and the Cepsilon promoter, whereas their dominant-negative counterparts decreased the ability of CD40 to activate NF-kappaB and the Cepsilon promoter. The overexpression of wild-type IKK-1 or IKK-2 seemed to cause toxic effects on the cells, whereas the dominant-negative forms were selective in their blockade of NF-kappaB and the Cepsilon promoter. These results suggest that CD40 employs TRAF-6, which presumably recruits NIK, which in turn employs IKK-1/IKK-2 to activate NF-kappaB and the Cepsilon promoter, the prologue to IgE switching. Thus the findings define a crucially important pathway in the generation of allergic states.

Summary Background Carbapenemase-producing Enterobacteriaceae (CPE) strains are encountered with increasing frequency in Europe. In November 2010 the European Centre for Disease Control (ECDC) graded Ireland as only having sporadic occurrence of CPE. Aim To describe the epidemiological and molecular typing analysis of the first outbreak of OXA-48-producing Klebsiella pneumoniae in an Irish tertiary care referral centre. Methods Sixteen OXA-48-producing K. pneumoniae isolates were detected, from both clinical and screening specimens, and analysed by pulsed-field gel electrophoresis and by multi-locus sequence typing. Findings Typing analysis revealed that two outbreak strains were circulating in the hospital, one among surgical patients and one among medical patients. The ‘medical strain' ST13 had already been identified as an internationally disseminated clone, whereas the ‘surgical strain' ST221 had not previously been reported as an OXA-48-carrying strain. Conclusion Although the outbreak on surgical wards was successfully controlled by implementing strict infection control measures, intermittent detection of individual patients carrying the ‘medical strain' of OXA-48 K. pneumoniae has persisted since then. The experience from this outbreak suggests that OXA-48 K. pneumoniae is endemic at low level in the healthcare setting in the Dublin region.

Five OXA-48-producing Klebsiella pneumoniae were detected in a tertiary referral hospital in Ireland between March and June 2011. They were found in the clinical isolates of five cases that were inpatients on general surgical wards. None of the cases had received healthcare at a facility outside of Ireland in the previous 12 months. This is the first report of OXA-48-producing K. pneumoniae in Ireland.

The role of the HU nucleoid-associated proteins in gene regulation was examined in Salmonella enterica serovar Typhimurium. The dimeric HU protein consists of different combinations of its α and β subunits. Transcriptomic analysis was performed with cultures growing at 37 °C at 1, 4 and 6 h after inoculation with mutants that lack combinations of HU α and HU β. Distinct but overlapping patterns of gene expression were detected at each time point for each of the three mutants, revealing not one but three regulons of genes controlled by the HU proteins. Mutations in the hup genes altered the expression of regulatory and structural genes in both the SPI1 and SPI2 pathogenicity islands. The hupA hupB double mutant was defective in invasion of epithelial cell lines and in its ability to survive in macrophages. The double mutant also had defective swarming activity and a competitive fitness disadvantage compared with the wild-type. In contrast, inactivation of just the hupB gene resulted in increased fitness and correlated with the upregulation of members of the RpoS regulon in exponential-phase cultures. Our data show that HU coordinates the expression of genes involved in central metabolism and virulence and contributes to the success of S. enterica as a pathogen.

The documentation of infection with meticillin-resistant Staphylococcus aureus (MRSA) on death certificates has been the subject of considerable public discussion. Using data from five tertiary referral hospitals in Ireland, we compared the documentation of MRSA and meticillin-susceptible S. aureus (MSSA) on death certificates in those patients who died in hospital within 30 days of having MRSA or MSSA isolated from blood cultures. A total of 133 patients had MRSA or MSSA isolated from blood cultures within 30 days of death during the study period. One patient was excluded as the death certificate information was not available; the other 132 patients were eligible for inclusion. MRSA and MSSA were isolated from blood cultures in 59 (44.4%) and 74 (55.6%) cases respectively. One patient was included as a case in both categories as both MRSA and MSSA were isolated from a blood culture. In 15 (25.4%) of the 59 MRSA cases, MRSA was documented on the death certificate. In nine (12.2%) of the 74 patients with MSSA cases, MSSA was documented on the death certificate. MRSA was more likely to be documented on the death certificate than MSSA (odds ratio: 2.46; 95% confidence interval: 1.01-6.01; P < 0.05). These findings indicate that there may be inconsistencies in the way organisms and infections are documented on death certificates in Ireland and that death certification data may underestimate the mortality related to certain organisms. In particular, there appears to be an overemphasis by certifiers on the documentation of MRSA compared with MSSA.

IgE switching requires the prior induction of C epsilon germline transcripts which is mediated by the concerted binding of STAT-6 and NF kappa B to the C epsilon promoter. These transcription factors are regulated by IL-4 and CD40, respectively. However the latter can effect other signaling pathways and the present study explores the role of p38 MAPK in induction of C epsilon germline transcripts. CD40 and IL-4, both alone and in synergy, were initially shown to activate the C epsilon promoter in a B cell lymphoma cell line. Under the same conditions CD40 caused activation of p38 MAPK, whereas IL-4 was ineffective. The p38 MAPK inhibitor, SB203580, and a dominant negative form of p38 MAPK decreased the CD40 activation of the C epsilon promoter by reducing the ability of CD40 to increase the transactivation potential of NF kappa B. This study suggests that p38 MAPK is crucially important in mediating CD40 activation of NF kappa B which acts to induce C epsilon germline transcripts, ultimately facilitating IgE switching.

We report the first documentation of C. difficile 027 in Ireland from a patient with diarrhoea transferred from a hospital in the United Kingdom (UK). In addition, we report two clusters of C. difficile ribotype 027 in two hospitals in Ireland.