Bacterial Microcompartment-Mediated Ethanolamine Metabolism in Escherichia coli Urinary Tract Infection.

Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic Escherichia coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal E. coli, both pathogenic enterohemorrhagic E. coli and commensal strains. During a UTI, urease-negative E. coli bacteria thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTIs is understudied. We evaluated the role of the metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analyzed infected urine samples by culture, high-performance liquid chromatography, reverse transcription-quantitative PCR, and genomic sequencing. The ethanolamine concentration in urine was comparable to the concentration of the most abundant reported urinary amino acid, d-serine. Transcription of the eut operon was detected in the majority of urine samples containing E. coli screened. All sequenced UPEC strains had conserved eut operons, while metabolic genotypes previously associated with UTI (dsdCXA, metE) were mainly limited to phylogroup B2. In vitro ethanolamine was found to be utilized as a sole source of nitrogen by UPEC strains. The metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly, eutE (which encodes acetaldehyde dehydrogenase) was required for UPEC strains to utilize ethanolamine to gain a growth advantage in AUM, suggesting that ethanolamine is also utilized as a carbon source. These data suggest that urinary ethanolamine is a significant additional carbon and nitrogen source for infecting E. coli strains.

Combined Analysis of Variation in Core, Accessory and Regulatory Genome Regions Provides a Super-Resolution View into the Evolution of Bacterial Populations.

The use of whole-genome phylogenetic analysis has revolutionized our understanding of the evolution and spread of many important bacterial pathogens due to the high resolution view it provides. However, the majority of such analyses do not consider the potential role of accessory genes when inferring evolutionary trajectories. Moreover, the recently discovered importance of the switching of gene regulatory elements suggests that an exhaustive analysis, combining information from core and accessory genes with regulatory elements could provide unparalleled detail of the evolution of a bacterial population. Here we demonstrate this principle by applying it to a worldwide multi-host sample of the important pathogenic E. coli lineage ST131. Our approach reveals the existence of multiple circulating subtypes of the major drug-resistant clade of ST131 and provides the first ever population level evidence of core genome substitutions in gene regulatory regions associated with the acquisition and maintenance of different accessory genome elements.

Synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement.

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV.

Antimicrobial susceptibility of long term care facility and general practice urine samples in patients 65 years and older: an observational study.

Background: Antimicrobial resistance in long-term care facilities (LTCFs) poses a risk to elderly residents. The aim of this observational study was to investigate recent patterns of antimicrobial susceptibility in urine samples submitted to the Microbiology Laboratory at Cork University Hospital (CUH) from LTCFs in the greater Cork region. The antimicrobial susceptibilities of LTCF and General Practitioner (GP) urine samples sent to CUH, for patients aged over 65 years of age, were compared. Methods: A retrospective analysis of the antimicrobial susceptibilities of urine samples submitted to the microbiology laboratory at CUH in quarter one of 2011-2014 was conducted. LTCF and GP urine sample susceptibilities, for patients over 65 years of age, were compared using Chi square statistics. Results: Overall, the LTCF urine samples were less susceptible than GP urine samples to the antimicrobials recommended in the national urinary tract infection guidelines; trimethoprim, nitrofurantoin, cephalexin, co-amoxiclav, ciprofloxacin and amoxicillin ( P < 0.001). Important trends in antimicrobial susceptibility over the time period were noted. A significant reduction in susceptibility to co-amoxiclav was found between Q1 2011 and Q1 2014 in both settings (GP P = 0.013, LTCF P = 0.005). This study provides important information which will contribute to the revision of antimicrobial prescribing guidelines in the future. Conclusion: This study highlights the need for continuous surveillance of antimicrobial susceptibility trends in LTCFs. Antimicrobial stewardship strategies are urgently required to address antimicrobial resistance and appropriate antimicrobial prescribing in the LTCF setting.

Parallel independent evolution of pathogenicity within the genus Yersinia.

The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.

Classification of polyhedral shapes from individual anisotropically resolved cryo-electron tomography reconstructions.

BACKGROUND: Cryo-electron tomography (cryo-ET) enables 3D imaging of macromolecular structures. Reconstructed cryo-ET images have a "missing wedge" of data loss due to limitations in rotation of the mounting stage. Most current approaches for structure determination improve cryo-ET resolution either by some form of sub-tomogram averaging or template matching, respectively precluding detection of shapes that vary across objects or are a priori unknown. Various macromolecular structures possess polyhedral structure. We propose a classification method for polyhedral shapes from incomplete individual cryo-ET reconstructions, based on topological features of an extracted polyhedral graph (PG). RESULTS: We outline a pipeline for extracting PG from 3-D cryo-ET reconstructions. For classification, we construct a reference library of regular polyhedra. Using geometric simulation, we construct a non-parametric estimate of the distribution of possible incomplete PGs. In studies with simulated data, a Bayes classifier constructed using these distributions has an average test set misclassification error of < 5 % with upto 30 % of the object missing, suggesting accurate polyhedral shape classification is possible from individual incomplete cryo-ET reconstructions. We also demonstrate how the method can be made robust to mis-specification of the PG using an SVM based classifier. The methodology is applied to cryo-ET reconstructions of 30 micro-compartments isolated from E. coli bacteria. CONCLUSIONS: The predicted shapes aren't unique, but all belong to the non-symmetric Johnson solid family, illustrating the potential of this approach to study variation in polyhedral macromolecular structures.

All Yersinia enterocolitica are pathogenic: virulence of phylogroup 1 Y. enterocolitica in a Galleria mellonella infection model.

Yersinia enterocolitica is a zoonotic pathogen and a common cause of gastroenteritis in humans. The species is composed of six diverse phylogroups, of which strains of phylogroup 1 are considered non-pathogenic to mammals due to the lack of the major virulence plasmid pYV, and their lack of virulence in a mouse infection model. In the present report we present data examining the pathogenicity of strains of Y. enterocolitica across all six phylogroups in a Galleria mellonellla model. We have demonstrated that in this model strains of phylogroup 1 exhibit severe pathogenesis with a lethal dose of as low as 10 c.f.u., that this virulence is an active process and that flagella play a major role in the virulence phenotype. We have also demonstrated that the complete lack of virulence in Galleria of the mammalian pathogenic phylogroups is not due to carriage of the pYV virulence plasmid. Our data suggest that all Y. enterocolitica can be pathogenic, which may be a reflection of the true natural habitat of the species, and that we may need to reconsider the eco-evo perspective of this important bacterial species.

Substrate channels revealed in the trimeric Lactobacillus reuteri bacterial microcompartment shell protein PduB.

Lactobacillus reuteri metabolizes two similar three-carbon molecules, 1,2-propanediol and glycerol, within closed polyhedral subcellular bacterial organelles called bacterial microcompartments (metabolosomes). The outer shell of the propanediol-utilization (Pdu) metabolosome is composed of hundreds of mainly hexagonal protein complexes made from six types of protein subunits that share similar domain structures. The structure of the bacterial microcompartment protein PduB has a tandem structural repeat within the subunit and assembles into a trimer with pseudo-hexagonal symmetry. This trimeric structure forms sheets in the crystal lattice and is able to fit within a polymeric sheet of the major shell component PduA to assemble a facet of the polyhedron. There are three pores within the trimer and these are formed between the tandem repeats within the subunits. The structure shows that each of these pores contains three glycerol molecules that interact with conserved residues, strongly suggesting that these subunit pores channel glycerol substrate into the metabolosome. In addition to the observation of glycerol occupying the subunit channels, the presence of glycerol on the molecular threefold symmetry axis suggests a role in locking closed the central region.

Yersinia enterocolitica: a brief review of the issues relating to the zoonotic pathogen, public health challenges, and the pork production chain.

Yersinia enterocolitica is a zoonotic agent that causes gastrointestinal disease in humans, as well as reactive arthritis and erythema nodosum. Enteropathogenic Yersinia are the etiological agents for yersiniosis, which can be acquired through the consumption of contaminated foods. As porcine animals are the main carriers of Y. enterocolitica, food safety measures to minimize human infection are of increasing interest to the scientific and medical community. In this review, we examine why it is imperative that information on the reservoirs, prevalence, virulence, and ability of this pathogen to survive in different environments is further investigated to provide rational measures to prevent or decrease associated disease risks.

Real-time Monitoring of Aerosol Generating Dental Procedures.

OBJECTIVE: We aimed to quantify aerosol concentrations produced during different dental procedures under different mitigation processes. METHOD: Aerosol concentrations were measured by the Optical Particle Sensor (OPS) and Wideband Integrated Bioaerosol Sensor (WIBS) during routine, time-recorded dental procedures on a manikin head in a partitioned enclosure. Four different, standardised dental procedures were repeated in triplicate for three different mitigation measures. RESULT: Both high-volume evacuation (HVE) and HVE plus local exhaust ventilation (LEV) eradicated all procedure-related aerosols, and the enclosure stopped procedure-related aerosols escaping. Aerosols recorded by the OPS and WIBS were 84 and 16-fold higher than background levels during tooth 16 FDI notation (UR6) drilling, and 11 and 24-fold higher during tooth 46 FDI notation (LR6) drilling, respectively. Ultrasonic scaling around the full lower arch (CL) or the full upper arch (CU) did not generate detectable aerosols with mitigation applied. Without mitigation the largest concentration of inhalable particles during procedures observed by the WIBS and OPS was during LR6 (139/cm3) and UR6 (28/cm3) drilling, respectively. Brief aerosol bursts were recorded during drilling procedures with HVE, these did not occur with LEV, suggesting LEV provides protection against operator errors. Variation was observed in necessary fallow times (49 - 280 minutes) without mitigation, while no particles remained airborne when mitigation was utilised. CONCLUSION: This data demonstrates that correctly positioned HVE or LEV is effective in preventing airborne spread and persistence of inhalable particles originating from dental AGPs. Additionally, a simple enclosure restricts the spread of aerosols outside of the operating area. CLINICAL SIGNIFICANCE: Employing correctly positioned HVE and LEV in non-mechanically ventilated clinics can prevent the dispersal and persistence of inhalable airborne particles during dental AGPs. Moreover, using enclosures have the additive effect of restricting aerosol spread outside of an operating area.

Stability of the Larsen B ice shelf on the Antarctic Peninsula during the Holocene epoch.

The stability of the Antarctic ice shelves in a warming climate has long been discussed, and the recent collapse of a significant part, over 12,500 km2 in area, of the Larsen ice shelf off the Antarctic Peninsula has led to a refocus toward the implications of ice shelf decay for the stability of Antarctica's grounded ice. Some smaller Antarctic ice shelves have undergone periodic growth and decay over the past 11,000 yr (refs 7-11), but these ice shelves are at the climatic limit of ice shelf viability and are therefore expected to respond rapidly to natural climate variability at century to millennial scales. Here we use records of diatoms, detrital material and geochemical parameters from six marine sediment cores in the vicinity of the Larsen ice shelf to demonstrate that the recent collapse of the Larsen B ice shelf is unprecedented during the Holocene. We infer from our oxygen isotope measurements in planktonic foraminifera that the Larsen B ice shelf has been thinning throughout the Holocene, and we suggest that the recent prolonged period of warming in the Antarctic Peninsula region, in combination with the long-term thinning, has led to collapse of the ice shelf.

Bacterial microcompartments and their role in pathogenicity.

Catabolic bacterial microcompartments (BMC), or metabolosomes, are self-assembling structures formed by enzymes enclosed by porous protein shells. They provide a specialised environment inside bacterial cells separating a short catabolic pathway with reactive or toxic intermediates from the cytoplasm. Substrates for microcompartment metabolism like ethanolamine and 1,2-propanediol are constantly produced in the human intestine by bacterial metabolism of food or host cell components. Enteric pathogens gain a competitive advantage in the intestine by metabolising these substrates, an advantage enhanced by the host inflammatory response. They exploit the intestinal specificity of signature metabolosome substrates by adopting substrate sensors and regulators encoded by BMC operons for governance of non-metabolic processes in pathogenesis. In turn, products of microcompartment metabolism regulate the host immune system.

Assessment of Environmental and Occupational Risk Factors for the Mitigation and Containment of a COVID-19 Outbreak in a Meat Processing Plant.

Throughout the COVID-19 pandemic, meat processing plants have been vulnerable to outbreaks of SARS-CoV-2 infection. Transmission of the virus is difficult to control in these settings because of a combination of factors including environmental conditions and the specific nature of the work. This paper describes a retrospective outbreak investigation in a meat processing plant, a description of the measures taken to prevent or contain further outbreaks, and insights on how those with specific knowledge of the working environment of these plants can collaborate with public health authorities to ensure optimal outbreak control. The plant experienced 111 confirmed positive asymptomatic cases in total with an estimated attack rate of 38% during a five-week period. 4 weeks after the first case, mass screening of all workers was conducted by the public health authorities. Thirty-two workers tested positive, of which 16 (50%) worked in one particular area of the plant, the boning hall (n = 60). The research team prepared and carried out semi-structured interviews with the plant personnel who were charged with COVID control within the plant. They carried out assessments of operational risk factors and also undertook air quality monitoring in the boning hall and abattoir. The air quality measurements in the boning hall showed a gradual build-up of carbon dioxide and aerosol particles over the course of a work shift, confirming that this poorly ventilated area of the plant had an environment that was highly favorable for aerosol transmission of SARS-CoV-2. Assessment of operational conditions incorporated visual surveys of the plant during the working day. Prior to and during the first 2 weeks of the outbreak, multiple measures were introduced into the plant by management, including physical distancing, provision of educational material to workers, visitor restrictions, and environmental monitoring. After the implementation of these measures and their progressive refinement by plant management, the factory had no further linked cases (clusters) or outbreaks for the following 198 days. The tailored approach to risk mitigation adopted in this meat processing plant shows that generic risk mitigation measures, as recommended by public health authorities, can be successfully adapted and optimized by designated plant emergency response teams.

Varicella Zoster Reactivation Causing Aseptic Meningitis in Healthy Adolescents: A Case Series And Review Of The Literature.

We describe 3 cases of adolescent varicella-zoster virus reactivation, complicated by aseptic meningitis, presenting to our institution in a 3-year period. These cases highlight varicella-zoster virus reactivation as an important cause of aseptic meningitis in the differential diagnosis of healthy adolescents, even in the absence of a characteristic exanthem. Evidence-based management recommendations are needed.

Tracking Yeast Metabolism and the Crabtree Effect in Real Time via CO2 Production using Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS).

In this study, a new approach to measure metabolic activity of yeast via the Crabtree effect is described. BARDS is an analytical technique developed to aid powder and tablet characterisation by monitoring changes in the compressibility of a solvent during solute dissolution. It is a rapid and simple method which utilises a magnetic stir bar to mix added solute and induce the acoustic resonance of a vessel containing a fixed volume of solvent. In this study it is shown that initiation of fermentation in a yeast suspension, in aqueous buffer, is accompanied by reproducible changes in the frequency of induced acoustic resonance. These changes signify increased compressibility of the suspension due to CO2 release by the yeast. A simple standardised BARDS protocol reveals yeast carbon source preferences and can generate quantitative kinetic data on carbon source metabolism which are characteristic of each yeast strain. The Crawford-Woods equation can be used to quantify total gaseous CO2 produced by a given number of viable yeast when supplied with a fixed amount of carbon source. This allows for a value to be calculated for the amount of gaseous CO2 produced by each yeast cell. The approach has the potential to transform the way in which yeast metabolism is tracked and potentially provide an orthogonal or surrogate method to determining viability, vitality and attenuation measurements in the future.

Effect of metabolosome encapsulation peptides on enzyme activity, coaggregation, incorporation, and bacterial microcompartment formation.

Metabolosomes, catabolic bacterial microcompartments (BMCs), are proteinaceous organelles that are associated with the breakdown of metabolites such as propanediol and ethanolamine. They are composed of an outer multicomponent protein shell that encases a specific metabolic pathway. Protein cargo found within BMCs is directed by the presence of an encapsulation peptide that appears to trigger aggregation before the formation of the outer shell. We investigated the effect of three distinct encapsulation peptides on foreign cargo in a recombinant BMC system. Our data demonstrate that these peptides cause variations in enzyme activity and protein aggregation. We observed that the level of protein aggregation generally correlates with the size of metabolosomes, while in the absence of cargo BMCs self-assemble into smaller compartments. The results agree with a flexible model for BMC formation based around the ability of the BMC shell to associate with an aggregate formed due to the interaction of encapsulation peptides.

The complete genome sequence and comparative genome analysis of the high pathogenicity Yersinia enterocolitica strain 8081.

The human enteropathogen, Yersinia enterocolitica, is a significant link in the range of Yersinia pathologies extending from mild gastroenteritis to bubonic plague. Comparison at the genomic level is a key step in our understanding of the genetic basis for this pathogenicity spectrum. Here we report the genome of Y. enterocolitica strain 8081 (serotype 0:8; biotype 1B) and extensive microarray data relating to the genetic diversity of the Y. enterocolitica species. Our analysis reveals that the genome of Y. enterocolitica strain 8081 is a patchwork of horizontally acquired genetic loci, including a plasticity zone of 199 kb containing an extraordinarily high density of virulence genes. Microarray analysis has provided insights into species-specific Y. enterocolitica gene functions and the intraspecies differences between the high, low, and nonpathogenic Y. enterocolitica biotypes. Through comparative genome sequence analysis we provide new information on the evolution of the Yersinia. We identify numerous loci that represent ancestral clusters of genes potentially important in enteric survival and pathogenesis, which have been lost or are in the process of being lost, in the other sequenced Yersinia lineages. Our analysis also highlights large metabolic operons in Y. enterocolitica that are absent in the related enteropathogen, Yersinia pseudotuberculosis, indicating major differences in niche and nutrients used within the mammalian gut. These include clusters directing, the production of hydrogenases, tetrathionate respiration, cobalamin synthesis, and propanediol utilisation. Along with ancestral gene clusters, the genome of Y. enterocolitica has revealed species-specific and enteropathogen-specific loci. This has provided important insights into the pathology of this bacterium and, more broadly, into the evolution of the genus. Moreover, wider investigations looking at the patterns of gene loss and gain in the Yersinia have highlighted common themes in the genome evolution of other human enteropathogens.

Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation.

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.

Mycobacterium bovis strains causing smear-positive human tuberculosis, Southwest Ireland.

Mycobacterium bovis caused 3% of human tuberculosis cases in southwest Ireland during 1998-2006. Of 11 M. bovis strains genotyped, 9 belonged to common animal spoligotypes. Seven strains were from sputum and potential sources of human-centered disease transmission. Ten-locus variable-number tandem repeat typing gave unique strain profiles and would detect disease outbreaks.

Plague.

Bubonic plague is an often fulminant systemic zoonosis, caused by Yersinia pestis. Conventional microbiology, bacterial population genetics, and genome sequence data, all suggest that Y pestis is a recently evolved clone of the enteric pathogen Yersinia pseudotuberculosis. The genetic basis of this organism's rapid adaptation to its insect vector (the flea) with transmission between mammalian hosts by novel subcutaneous and pneumonic routes of infection is becoming clearer. This transition provides a paradigm for the way in which new pathogens could emerge. Plague in humans is controlled by suppression of rodent reservoir hosts and their fleas and by early detection and treatment of cases of disease. Detection systems for plague in non-endemic regions might now be needed because of a bioterrorism threat. Rapid diagnostic tests are available and a subunit vaccine is in clinical trials.