A Large, Refractory Nosocomial Outbreak of Klebsiella pneumoniae Carbapenemase-Producing Escherichia coli Demonstrates Carbapenemase Gene Outbreaks Involving Sink Sites Require Novel Approaches to Infection Control.

Carbapenem-resistant Enterobacteriaceae (CRE) represent a health threat, but effective control interventions remain unclear. Hospital wastewater sites are increasingly being highlighted as important potential reservoirs. We investigated a large Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli outbreak and wider CRE incidence trends in the Central Manchester University Hospital NHS Foundation Trust (CMFT) (United Kingdom) over 8 years, to determine the impact of infection prevention and control measures. Bacteriology and patient administration data (2009 to 2017) were linked, and a subset of CMFT or regional hospital KPC-producing E. coli isolates (n = 268) were sequenced. Control interventions followed international guidelines and included cohorting, rectal screening (n = 184,539 screens), environmental sampling, enhanced cleaning, and ward closure and plumbing replacement. Segmented regression of time trends for CRE detections was used to evaluate the impact of interventions on CRE incidence. Genomic analysis (n = 268 isolates) identified the spread of a KPC-producing E. coli outbreak clone (strain A, sequence type 216 [ST216]; n = 125) among patients and in the environment, particularly on 2 cardiac wards (wards 3 and 4), despite control measures. ST216 strain A had caused an antecedent outbreak and shared its KPC plasmids with other E. coli lineages and Enterobacteriaceae species. CRE acquisition incidence declined after closure of wards 3 and 4 and plumbing replacement, suggesting an environmental contribution. However, ward 3/ward 4 wastewater sites were rapidly recolonized with CRE and patient CRE acquisitions recurred, albeit at lower rates. Patient relocation and plumbing replacement were associated with control of a clonal KPC-producing E. coli outbreak; however, environmental contamination with CRE and patient CRE acquisitions recurred rapidly following this intervention. The large numbers of cases and the persistence of blaKPC in E. coli, including pathogenic lineages, are of concern.

Predicting antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data.

OBJECTIVES: Whole-genome sequencing potentially represents a single, rapid and cost-effective approach to defining resistance mechanisms and predicting phenotype, and strain type, for both clinical and epidemiological purposes. This retrospective study aimed to determine the efficacy of whole genome-based antimicrobial resistance prediction in clinical isolates of Escherichia coli and Klebsiella pneumoniae. METHODS: Seventy-four E. coli and 69 K. pneumoniae bacteraemia isolates from Oxfordshire, UK, were sequenced (Illumina HiSeq 2000). Resistance phenotypes were predicted from genomic sequences using BLASTn-based comparisons of de novo-assembled contigs with a study database of >100 known resistance-associated loci, including plasmid-associated and chromosomal genes. Predictions were made for seven commonly used antimicrobials: amoxicillin, co-amoxiclav, ceftriaxone, ceftazidime, ciprofloxacin, gentamicin and meropenem. Comparisons were made with phenotypic results obtained in duplicate by broth dilution (BD Phoenix). Discrepancies, either between duplicate BD Phoenix results or between genotype and phenotype, were resolved with gradient diffusion analyses. RESULTS: A wide variety of antimicrobial resistance genes were identified, including blaCTX-M, blaLEN, blaOKP, blaOXA, blaSHV, blaTEM, aac(3')-Ia, aac-(3')-IId, aac-(3')-IIe, aac(6')-Ib-cr, aadA1a, aadA4, aadA5, aadA16, aph(6')-Id, aph(3')-Ia, qnrB and qnrS, as well as resistance-associated mutations in chromosomal gyrA and parC genes. The sensitivity of genome-based resistance prediction across all antibiotics for both species was 0.96 (95% CI: 0.94-0.98) and the specificity was 0.97 (95% CI: 0.95-0.98). Very major and major error rates were 1.2% and 2.1%, respectively. CONCLUSIONS: Our method was as sensitive and specific as routinely deployed phenotypic methods. Validation against larger datasets and formal assessments of cost and turnaround time in a routine laboratory setting are warranted.

'Feral' and 'wild'-type methicillin-resistant Staphylococcus aureus in the United Kingdom.

Circulation of methicillin-resistant Staphylococcus aureus (MRSA) outside hospitals could alter the impact of hospital-based control strategies. We investigated two groups of cases (each matched to controls with MRSA): 61 'community cases' not in acute hospital in the year before MRSA isolation; and 21 cases with ciprofloxacin-sensitive (CipS) MRSA. Multi-locus sequence typing, spa-typing and Panton-Valentine leukocidin gene testing were performed and demographics obtained. Additional questionnaires were completed by community case GPs. Community cases comprised 6% of Oxfordshire MRSA. Three community cases had received no regular healthcare or antibiotics: one was infected with CipS. Ninety-one percent of community cases had healthcare-associated sequence type (ST)22/36; CipS MRSA cases had heterogeneous STs but many had recent healthcare exposure. A substantial minority of UK MRSA transmission may occur outside hospitals. Hospital strains are becoming 'feral' or persisting in long-term carriers in the community with regular healthcare contacts; those with recent healthcare exposure may nevertheless acquire non-hospital epidemic MRSA strains in the community.

In developing hippocampal neurons, NR2B-containing N-methyl-D-aspartate receptors (NMDARs) can mediate signaling to neuronal survival and synaptic potentiation, as well as neuronal death.

It has been suggested that NR2B-containing N-methyl-d-aspartate (NMDA) receptors have a selective tendency to promote pro-death signaling and synaptic depression, compared with the survival promoting, synapse potentiating properties of NR2A-containing NMDA receptors. A preferential localization of NR2A-containing NMDA receptors at the synapse in maturing neurons could thus explain differences in synaptic vs. extrasynaptic NMDA receptor signaling. We have investigated whether NMDA receptors can mediate signaling to survival, death, and synaptic potentiation, in dissociated rat neuronal cultures at a developmental stage prior to significant NR2A expression and subunit-specific differences between synaptic and extrasynaptic NMDA receptors. We show that in developing hippocampal neurons, the progressive reduction in sensitivity of NMDA receptor currents to the NR2B antagonist ifenprodil applies to both synaptic and extrasynaptic locations. However, the reduction is less acute in extrasynaptic currents, indicating that NR2A does partition preferentially, but not exclusively, into synaptic locations at DIV>12. We then studied NMDA receptor signaling at DIV10, when both synaptic and extrasynaptic NMDA receptors are both overwhelmingly and equally NR2B-dominated. To analyze pro-survival signaling we studied the influence of synaptic NMDA receptor activity on staurosporine-induced apoptosis. Blockade of spontaneous NMDAR activity with MK-801, or ifenprodil exacerbated the apoptotic insult. Furthermore, MK-801 and ifenprodil both antagonized neuroprotection promoted by enhancing synaptic activity. Pro-death signaling induced by a toxic dose of NMDA is also blocked by NR2B-specific antagonists. Using a cell culture model of synaptic NMDA receptor-dependent synaptic potentiation, we find that this is mediated exclusively by NR2B-containing N-methyl-D-aspartate receptors, as implicated by NR2B-specific antagonists and the use of selective vs. non-selective doses of the NR2A-preferring antagonist NVP-AAM077. Therefore, within a single neuron, NR2B-NMDA receptors are able to mediate both survival and death signaling, as well as model of NMDA receptor-dependent synaptic potentiation. In this instance, subunit differences cannot account for the dichotomous nature of NMDA receptor signaling.

A role for protein kinases and phosphatases in the Ca(2+)-induced enhancement of hippocampal AMPA receptor-mediated synaptic responses.

We have investigated the effects of inhibitors of protein kinases and protein phosphatases on the NMDA receptor-independent potentiation of evoked and miniature (m) excitatory postsynaptic currents (EPSCs) induced by the entry of Ca2+ via voltage-gated Ca2+ channels in hippocampal CA1 pyramidal neurons. Voltage pulse-induced potentiation was markedly attenuated when evoked in the presence of the protein kinase blockers KN-62, K-252a, or H-7. Bath application of the protein phosphatase inhibitor calyculin A converted the usual transient potentiation of both evoked and spontaneous EPSCs induced by voltage pulses into a more sustained potentiation. Similarly, the introduction of the phosphatase inhibitors microcystin LR or okadaic acid into postsynaptic cells, via patch pipettes, also resulted in a sustained increase in the amplitude of mEPSCs. We propose that entry of Ca2+ into CA1 neurons activates calcium/calmodulin-dependent protein kinase II, which leads to an enhanced responsiveness of synaptic AMPA receptor channels. The enhancement is transient, however, owing to postsynaptic phosphatase activity.

A rise in postsynaptic Ca2+ potentiates miniature excitatory postsynaptic currents and AMPA responses in hippocampal neurons.

We have investigated the site of expression of the potentiation of excitatory postsynaptic currents (EPSCs) induced by the activation of postsynaptic voltage-sensitive Ca2+ channels, by examining the effect of depolarizing pulses on miniature (m) EPSCs and responses to AMPA. Application of voltage pulses caused a approximately 2.5-fold increase in the mean amplitude of mEPSCs. This NMDA receptor-independent potentiation of mEPSC amplitudes was transient, returning to control values within 30-40 min. The potentiation was associated with a decrease in the number of small amplitude events and an increase in the number, as well as the maximum amplitude, of the larger events, with no apparent change in mEPSC kinetics. Accompanying the increase in mEPSC amplitudes, there was a 1.6-fold increase in the apparent frequency of events. Voltage pulse-induced potentiation was completely blocked by the inclusion of the Ca2+ chelator BAPTA in the recording pipette. Responses to repeated applications of AMPA were also potentiated following the application of voltage pulses, and the time course of this potentiation was similar to that observed with the mEPSCs. Our data indicate that rises in intracellular Ca2+ that occur independently of NMDA receptor activation can result in a potentiation of quantal size, which is due to an increase in the postsynaptic sensitivity of non-NMDA receptors.

Taking the time to study competitive antagonism.

Selective receptor antagonists are one of the most powerful resources in a pharmacologist's toolkit and are essential for the identification and classification of receptor subtypes and dissecting their roles in normal and abnormal body function. However, when the actions of antagonists are measured inappropriately and misleading results are reported, confusion and wrong interpretations ensue. This article gives a general overview of Schild analysis and the method of determining antagonist equilibrium constants. We demonstrate why this technique is preferable in the study of competitive receptor antagonism than the calculation of antagonist concentration that inhibit agonist-evoked responses by 50%. In addition we show how the use of Schild analysis can provide information on the outcome of single amino acid mutations in structure-function studies of receptors. Finally, we illustrate the need for caution when studying the effects of potent antagonists on synaptic transmission where the timescale of events under investigation is such that ligands and receptors never reach steady-state occupancy.

Late-phase, protein synthesis-dependent long-term potentiation in hippocampal CA1 pyramidal neurones with destabilized microtubule networks.

BACKGROUND AND PURPOSE: Protein synthesis-dependent late-long term potentiation (L-LTP) is an enduring form of synaptic plasticity that has been shown to rely on, at least partly, protein synthesis at synaptic and/or dendritic sites. Evidence suggests that somatic transcription of new mRNAs may provide a significant contribution to the availability of mRNAs at synaptic sites where they are made available for dendritic translation. Transport of mRNAs from somatic to dendritic sites might be expected to involve movement along a microtubule network. In this study we examined whether it was possible to maintain L-LTP in hippocampal slices with destabilized microtubule networks. EXPERIMENTAL APPROACH: Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from rat hippocampal slices and following a period of baseline recording, stimuli were given that induced LTP. LTP was monitored for 5 h in both control slices and slices treated with vincristine to depolymerize tubulin. KEY RESULTS: L-LTP was induced and maintained in vincristine-treated slices. Four hours after tetanic stimulation fEPSPs were 196+/-19% of baseline values. The magnitude of potentiation was similar to that seen in untreated slices (175+/-15%). L-LTP in vincristine-treated slices was, however, not maintained in the presence of the protein synthesis inhibitor, rapamycin. Immunohistochemistry and confocal microscopy of vincristine-treated slices verified that the microtubule network had been destabilized. CONCLUSIONS AND IMPLICATIONS: Communication between somatic and synaptic sites through protein and/or mRNA trafficking via an intact microtubule network is not required for protein synthesis dependent L-LTP.

Hospital exposure in a UK population, and its association with bacteraemia.

Despite the importance of healthcare-associated infection, few studies have quantified the association between severe infection and hospital exposure in UK populations. Our aim was to estimate the proportion of the population with recent hospital admission, together with rates of infection in hospital-exposed and hospital-naïve populations. We studied bacteraemia as a marker of severe infection in a population of 550,000, served by two hospitals, between 1 April 2000 and 31 March 2005. Hospital-exposed persons accounted for 8.3% of the population, defined as having been resident in a hospital in the last year. The hospital-exposed population accounted for 55% of all admissions, and 42% of emergency admissions to medical, paediatric or surgery departments. After adjustment for age, the hospital-exposed group had much higher rates of admission bacteraemia. Age-standardised incidence rate ratios relative to hospital-naïve patients were 43 [95% confidence interval (CI): 22-85] for meticillin-resistant Staphylococcus aureus (MRSA), 20 (15-27) for S. aureus other than MRSA, 7.3 (5.2-10) for Streptococcus pneumoniae, and 14 (11-18) for E. coli. MRSA was common among hospital-exposed admissions, including emergencies in hospital-exposed men, rates of admission MRSA bacteraemia (31 per 100,000 per annum) and S. pneumoniae bacteraemia (33 per 100,000 per annum) were similar. This quantitative analysis confirms that prior hospital admission is a major risk factor for bacteraemia on hospital admission; it is unclear whether acquisition of pathogens in hospital, co-morbidity or other factors explain this.

Bacteraemia prediction in emergency medical admissions: role of C reactive protein.

AIM: To define the contribution made by C reactive protein (CRP) measurement to bacteraemia prediction in adults with medical emergencies in the UK. METHODS: This two year cohort study involved 6234 patients admitted as emergency cases to the acute medical or infectious diseases services of the Oxford Radcliffe Hospitals, in whom blood cultures were taken on arrival. The main outcome measures were bacteraemia risk associated with admission CRP concentrations, lymphocyte counts, and neutrophil counts. RESULTS: The quantitative associations between CRP concentration, admission lymphocyte count, and neutrophil count were defined. Risk of bacteraemia rose continuously as the CRP increased: no "cutoff" value was evident. Models examining combinations of CRP, neutrophil count, and lymphocyte count were developed and validated using a split sample technique. CRP contributed to a model including lymphocyte and neutrophil counts, but its effect was small. CRP alone performed no better than either a model combining lymphopenia and neutrophilia, or than lymphopenia alone. CONCLUSIONS: In patients with acute medical emergencies who are suspected of bacteraemia clinically, CRP concentrations, although associated with bacteraemia, have a limited role in bacteraemia prediction.

Role of data warehousing in healthcare epidemiology.

Electronic storage of healthcare data, including individual-level risk factors for both infectious and other diseases, is increasing. These data can be integrated at hospital, regional and national levels. Data sources that contain risk factor and outcome information for a wide range of conditions offer the potential for efficient epidemiological analysis of multiple diseases. Opportunities may also arise for monitoring healthcare processes. Integrating diverse data sources presents epidemiological, practical, and ethical challenges. For example, diagnostic criteria, outcome definitions, and ascertainment methods may differ across the data sources. Data volumes may be very large, requiring sophisticated computing technology. Given the large populations involved, perhaps the most challenging aspect is how informed consent can be obtained for the development of integrated databases, particularly when it is not easy to demonstrate their potential. In this article, we discuss some of the ups and downs of recent projects as well as the potential of data warehousing for antimicrobial resistance monitoring.

Polysymptomatic complaints and Briquet's syndrome in polycystic ovary disease.

To test the hypothesis that there is an association between polycystic ovary disease and Briquet's syndrome, the authors administered a health questionnaire to infertile women with polycystic ovary disease, infertile women with tubal disease, and normal women. The patients with polycystic ovary disease endorsed significantly more physical and psychological complaints than either control group. Structured interviews revealed that five of the 39 (13%) met diagnostic criteria for definite or probable Briquet's syndrome. This study gives support to an association between polysymptomatic complaints, Briquet's syndrome, and polycystic ovary disease.

A novel mammalian expression screen exploiting green fluorescent protein-based transcription detection in single cells.

The accumulation of DNA sequence information from large-scale genomic and random library sequencing projects is leading to the rapid identification of many putative genes, virtual transcripts and ESTs of unknown function. There is therefore an increasing need for high throughput, sensitive and robust methods for identification and characterisation of genes, and/or their products, based on function. We describe a high throughput functional expression screen based on semi-quantitative analysis of enhanced green fluorescent protein expression in single cells by confocal microscopy. The assay was implemented in a micro-scale format, requiring around 10(4) cells/test. The system was validated by co-transfection of a series of cDNAs encoding pro-inflammatory cytokine intracellular signal mediators with a d2EGFP reporter containing a cytokine responsive promoter. The majority of the test plasmids gave a detectable signal above background at a pool size of 250-500. Replicate tests indicate that the assay is reproducible at this pool size. At this level we demonstrate that large (>10(6) transformants) libraries can be feasibly screened.

Single-channel currents from recombinant NMDA NR1a/NR2D receptors expressed in Xenopus oocytes.

We have investigated the single-channel and whole-cell behaviour of recombinant N-methyl-D-aspartate (NMDA) receptors formed from NR1a and NR2D receptor subunits expressed in Xenopus oocytes. The EC50 for apparent steady-state activation of NR1a/NR2D receptors by glutamate was 450 nM, while extracellular MG2+ produced a voltage-dependent block of glutamate responses with an IC50 of 440 microM at -70 mV. At negative holding potentials glutamate-activated NR1a/NR2D single-channel currents, in 0.85 mM external Ca2+, had slope conductances of 35 pS for the main level, and 17 pS for the sublevel; direct transitions occurred between these two conductance levels. On average 35 pS events had mean open times of 1.01 +/- 0.04 ms, whereas the mean open times of 17 pS events were consistently longer (1.28 +/- 0.06 ms). In 5 mM external Ca2+ the larger conductance level was reduced to 20 pS whereas in Ca(2+)-free solutions it was increased to 50 pS. The frequency of transitions between the main and subconductance levels showed temporal asymmetry: 35-17 pS transitions were more frequent (61%) than 17-35 pS transitions. This asymmetry was not affected by alterations in the external Ca2+ concentration (up to 5 mM). In conclusion, the NR1a/NR2D channel is, like NR1a/NR2C, a 'low conductance' NMDA channel, but it can be distinguished from NR1a/NR2C channels on the basis of transition asymmetry and differences in the open times of its main and sub-conductance levels.

Determination of NMDA NR1 subunit copy number in recombinant NMDA receptors.

Co-expression of wild-type and mutated NMDA NR1 (N598R) subunits in Xenopus oocytes has been used to determine the stoichiometry of the NMDA receptor-channel. When expressed together, wild-type NR2A and mutant NR1 (N598R) subunits produced channels with a main conductance of 2.6 pS and a sublevel of 1.2 pS. These conductances were clearly different from those obtained from wild-type NR1 and wild-type NR2A channels which gave characteristic 50 pS events with a 40 pS sublevel. When wild-type and mutant NR1 subunits were co-expressed together with NR2A subunits a different channel type with a main conductance of 15.2 pS and a sublevel of 11.4 pS was obtained, as well as the 'all wild-type' and 'all mutant' channels described above. These results indicate that there are likely to be two copies of the NR1 subunit in each NMDA receptor complex.

Relation between lymphopenia and bacteraemia in UK adults with medical emergencies.

AIMS: To determine the relevance of lymphopenia to the diagnosis of bacteraemia in patients admitted with medical emergencies, relative to peripheral blood white cell count and neutrophilia. PATIENTS/METHODS: A two year cohort study carried out in a teaching hospital in Oxford, UK of 21,495 consecutive adult emergency admissions to general medical or infectious disease wards. Full blood data were available in 21,372 cases; 41 cases with extreme full blood count results (neutrophil count, > 75 x 10(9)/litre; lymphocyte count, > 10 x 10(9)/litre) were excluded, leaving 21,331 cases for analysis. The association between the admission lymphocyte and neutrophil counts and the risk of bacteraemia was assessed. RESULTS: Neutrophilia and lymphopenia were both associated with bacteraemia. Lymphopenia was the better predictor in this cohort. Both neutrophilia and lymphopenia were more predictive of bacteraemia than the total white blood cell count. CONCLUSIONS: Both lymphocyte and neutrophil counts, rather than total white blood cell count, should be considered in adult medical admissions with suspected bacteraemia.

Measuring change in surgical practice.

We reviewed the work of a single general surgical firm over six years--about 11,500 patient episodes. Although the workload remained approximately constant, a severe reduction in the number of available beds was accompanied by a marked change towards shorter duration of stay. This affected particularly the elective cases. Previous authors, when trying to predict requirement of hospital beds, have accepted that duration of stay and number of beds available are independent variables; this was not what we observed.

Pursuing efficiency in surgical practice.

To examine fluctuations in numbers of patients on surgical wards the dates of admission from January of each of the 5556 patients admitted from 1 January 1985 to 31 December 1987 were examined during computerised audit of a single surgical firm. The numbers of patients under the care of the firm fluctuated widely, often exceeding the 38 beds nominally available. Duration of stay varied from two days or less (3062 admissions) to more than a month (163 admissions). One patient was in hospital for 278 days. The patients admitted for more than a month (2.9% of the total) filled 28% of the beds; not all these patients were elderly. A further increase in throughput of patients undergoing elective operations might be achieved by always admitting patients on the day of operation, and perhaps by discharging patients even sooner than at present. Efficiency would increase but so would overall costs.

Healthcare-associated outbreak of meticillin-resistant Staphylococcus aureus bacteraemia: role of a cryptic variant of an epidemic clone.

BACKGROUND: New strains of meticillin-resistant Staphylococcus aureus (MRSA) may be associated with changes in rates of disease or clinical presentation. Conventional typing techniques may not detect new clonal variants that underlie changes in epidemiology or clinical phenotype. AIM: To investigate the role of clonal variants of MRSA in an outbreak of MRSA bacteraemia at a hospital in England. METHODS: Bacteraemia isolates of the major UK lineages (EMRSA-15 and -16) from before and after the outbreak were analysed by whole-genome sequencing in the context of epidemiological and clinical data. For comparison, EMRSA-15 and -16 isolates from another hospital in England were sequenced. A clonal variant of EMRSA-16 was identified at the outbreak hospital and a molecular signature test designed to distinguish variant isolates among further EMRSA-16 strains. FINDINGS: By whole-genome sequencing, EMRSA-16 isolates during the outbreak showed strikingly low genetic diversity (P < 1 × 10(-6), Monte Carlo test), compared with EMRSA-15 and EMRSA-16 isolates from before the outbreak or the comparator hospital, demonstrating the emergence of a clonal variant. The variant was indistinguishable from the ancestral strain by conventional typing. This clonal variant accounted for 64/72 (89%) of EMRSA-16 bacteraemia isolates at the outbreak hospital from 2006. CONCLUSIONS: Evolutionary changes in epidemic MRSA strains not detected by conventional typing may be associated with changes in disease epidemiology. Rapid and affordable technologies for whole-genome sequencing are becoming available with the potential to identify and track the emergence of variants of highly clonal organisms.

Influence of GluN2 subunit identity on NMDA receptor function.

N-methyl-d-aspartate receptors (NMDARs) are ligand-gated ion channels ('ionotropic' receptors) activated by the major excitatory neurotransmitter, l-glutamate. While the term 'the NMDAR' is often used it obscures the fact that this class of receptor contains within it members whose properties are as different as they are similar. This heterogeneity was evident from early electrophysiological, pharmacological and biochemical assessments of the functional properties of NMDARs and while the molecular basis of this heterogeneity has taken many years to elucidate, it indicated from the outset that the diversity of NMDAR phenotypes could allow this receptor family to subserve a variety of functions in the mammalian central nervous system. In this review we highlight some recent studies that have identified structural elements within GluN2 subunits that contribute to the heterogeneous biophysical properties of NMDARs, consider why some recently described novel pharmacological tools may permit better identification of native NMDAR subtypes, examine the evidence that NMDAR subtypes differentially contribute to the induction of long-term potentiation and long-term depression and discuss how through the use of chimeric proteins additional insights have been obtained that account for NMDAR subtype-dependency of physiological and pathophysiological signalling. This article is part of the Special Issue entitled 'Glutamate Receptor-Dependent Synaptic Plasticity'.