Pseudomonas aeruginosa PcrV and Psl, the Molecular Targets of Bispecific Antibody MEDI3902, Are Conserved Among Diverse Global Clinical Isolates.

Background: Bispecific antibody MEDI3902, targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV) and Psl exopolysaccharide, is currently in phase 2b development for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation. We surveyed a diverse collection of isolates to study MEDI3902 epitope conservation and protective activity. Methods: P. aeruginosa clinical isolates (n = 913) were collected from diverse patients and geographic locations during 2003-2014. We conducted whole-genome sequencing; performed PcrV and Psl expression analyses via immunoblotting and enzyme-linked immunosorbent assay, respectively; performed crystallography to determine the MEDI3902 PcrV epitope, using anti-PcrV Fab and PcrV components (resolved at 2.8 Å); and evaluated MEDI3902 protective activity against select isolates in vitro and in vivo. Results: Intact psl operon and pcrV genes were present in 94% and 99% of isolates, respectively, and 99.9% of isolates contained at least one of the genetic elements. Anti-Psl binding was confirmed in tested isolates harboring a complete Psl operon or lacking nonessential psl genes. We identified 46 PcrV variant sequences, and MEDI3902-PcrV contact residues were preserved. MEDI3902 maintained potent in vivo activity against various strains, including strains expressing only a single target. Conclusions: Psl and PcrV are highly prevalent in global clinical isolates, suggesting MEDI3902 can mediate broad coverage against P. aeruginosa.

Avibactam and class C ß-lactamases: mechanism of inhibition, conservation of the binding pocket, and implications for resistance.

Avibactam is a novel non-ß-lactam ß-lactamase inhibitor that inhibits a wide range of ß-lactamases. These include class A, class C, and some class D enzymes, which erode the activity of ß-lactam drugs in multidrug-resistant pathogens like Pseudomonas aeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the ß-lactam antibiotics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first ß-lactamase inhibitor that might provide activity against class C-mediated resistance, which represents a growing concern in both hospital- and community-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, but in contrast to other currently used ß-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution structure of avibactam bound to a class C ß-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, the structural information was used to predict effective inhibition by sequence analysis and functional studies of class C ß-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtained from recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam.

Relationship between Trendelenburg tilt and internal jugular vein diameter.

OBJECTIVES: To evaluate the relationship between Trendelenburg tilt and internal jugular vein (IJV) diameter, and to examine any cumulative effects of tilt on the IJV diameter. METHODS: Using a tilt table, healthy volunteers were randomised to Trendelenburg tilts of 10 degrees, 15 degrees, 20 degrees, 25 degrees, and 30 degrees. Ultrasound was used to measure and record the lateral diameter of the right IJV at the level of the cricoid cartilage. Following each reading the table was returned to the supine position. Balanced randomisation was used to assess cumulative tilt effects. RESULTS: A total of 20 healthy volunteers were recruited (10 men, 10 women). Mean supine IJV diameter was 13.5 mm (95% CI 12.8 to 14.1) and was significantly greater at 10 degrees (15.5 mm, 95% CI 14.9 to 16.1). There was no significant difference between 10 degrees and greater angles of tilt. The effect of the previous angle of tilt did not prove to be statistically significant. CONCLUSION: Increasing the degree of Trendelenburg tilt increases the lateral diameter of the IJV. Even a 10 degrees tilt is effective. The cumulative effect of tilt (that is, the effect of the previous angle) is not significant. Ultrasound guided cannulation is ideal, but in its absence Trendelenburg tilt will increase IJV diameter and improve the chance of successful cannulation. While 25 degrees achieved optimum distension, this may not be practical and may be detrimental (for example, risk of raised intracranial pressure).

Survey of attitudes of senior emergency physicians towards the introduction of emergency department ultrasound.

INTRODUCTION: Emergency department ultrasound (EDU) is widely practised in the USA, Australia, parts of Europe, and Asia. EDU has been used in the UK since the late 1990s but as yet, few areas have established a practice. OBJECTIVES: To assess the current climate of opinion with respect to the practice, constraints, and establishment of EDU among emergency department (ED) consultants on the island of Ireland. METHODS: A postal questionnaire was formulated, piloted, and assessed for ambiguity by a sample of ED consultants and an independent non-ED consultant, prior to being mailed to all ED consultants in Ireland. RESULTS: Of the 58 consultants canvassed 46 (79%) responded. Of the respondents, 40 (87%) strongly agreed/agreed that EDU is appropriate and should be performed in the ED. Of these, 3 (7%) are currently performing EDU; 37 (80%) have not had formal training in EDU, however 42 (91%) support the establishment of national guidelines for training in focused ultrasound in the ED. Problems instituting EDU were often multifactorial. Commonly highlighted difficulties included financial issues (24 respondents, 52%) and radiology department support (16 respondents, 34%). Other cited problems include varying interdepartmental practices (15 respondents, 33%) and (for some EDs) low numbers of patients requiring EDU, with projected difficulties in skills maintenance. CONCLUSION: Despite the vast majority of ED consultants being in favour of EDU, very few actually perform it on a regular basis or have had any formal training. Highlighted difficulties in EDU implementation included financial constraints, lack of support from radiology departments, and lack of formal training.

Non-touch suturing technique fails to reduce glove puncture rates in an accident and emergency department.

OBJECTIVE: To assess the impact of introducing a safer non-touch suturing technique into an inner city emergency department. METHODS: The rate of glove perforation, measured by electrical conductance, was used as a marker. Gloves (Bodyguards) used in suturing were collected over a two month period. Two half day suture workshops were then conducted in the emergency department and gloves were collected for a further two months. All doctors (14) were included in the study regardless of grade. Gloves were tested for perforation by electrical conductance. RESULTS: There were 107 gloves and 19 perforations in the first group, 133 with 28 perforations in the second (p = 0.52), and one perforation in 100 control gloves. Most doctors reported inadvertent needlestick handling. CONCLUSION: Two half day suturing workshops are not enough to reduce glove perforations from suturing in the emergency department. Difficulty with compliance suggests that safe suturing practice must be taught before doctors develop bad habits that are difficult to change.

Are we getting the point? Needlestick injuries-an ongoing problem.

The pattern of glove perforation using hollow needles is unknown and the incidence, using sensitive electronic methods has not been described. We wished to identify the glove perforation rate following the use of hollow needles in our Emergency Department. Two hundred and eighty-nine gloves were collected over three months. These gloves were electronically tested and compared with one hundred unused control gloves. There were 28/298 perforations (9.7%) vs 1/100 in the control group (P<0.01 Fisher's exact probability). Most perforations were located on the thumb and index fingers. There were no needlestick injuries reported during this period. The glove perforation rate following hollow needle use in our Emergency Department is higher than previously reported. The pattern of perforation suggests injury by the needle held in the dominant hand.

Ionising radiation (medical exposure) regulations (Northern Ireland) 2000 and their implications for Accident and Emergency (A&E) doctors in training.

With the introduction of the Ionising Radiation (Medical Exposure) Regulations (Northern Ireland) 2000 (IRMER) the medical practitioner faces greater accountability when requesting radiological investigations. The referrer (usually a doctor or dentist) must supply sufficient medical data to justify radiation exposure to a patient. These regulations can lead to criminal prosecution if breached. Our objectives were to identify the level of unjustified requests for plain abdominal radiography among A&E doctors and whether there is a statistically significant difference in the justification of request between doctors of differing experience. We reviewed and prepared statistical analysis of 100 A&E request forms for plain abdominal radiography. Royal College of Radiologist Guidelines were used as a "Gold standard" for justification of the investigation. A&E doctors of less than six months experience are at greater risk of breaching these regulations when requesting plain abdominal films, when compared to more experienced doctors. This is a serious issue which should be addressed at undergraduate and pre-registration level in addition to ongoing audit.

Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus.

Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.

Nucleotide sequence of the streptococcin A-FF22 lantibiotic regulon: model for production of the lantibiotic SA-FF22 by strains of Streptococcus pyogenes.

Streptococcin A-FF22 (SA-FF22) is a type AII linear lantibiotic produced by Streptococcus pyogenes strain FF22. Sequence analysis of an approximate 10 kb region of DNA showed it to contain nine open reading frames arranged in three operons responsible for regulation, biosynthesis and immunity of SA-FF22. This region is organized similarly to the Lactococcus lactis lacticin 481 region, however, unlike lacticin 481, a two-component regulatory system is essential for SA-FF22 production. Located immediately downstream of the scn region is a putative transposase gene, the presence of which supports earlier data that indicated a mobile nature to this region.

Effect of inactivation of gtf genes on adherence of Streptococcus downei.

The activity of glucosyltransferases (GTF), a group of enzymes that synthesize water-soluble and -insoluble glucans from sucrose, significantly contributes to the cariogenicity of mutans streptococci. Streptococcus downei produces four glucosyltransferases, GTFI, which produces insoluble glucan, and GTFS, GTFT, and GTFU, which synthesize soluble glucans. We have previously reported that inactivation of gtfS results in altered adherence and have now examined its interaction with other enzymes by constructing mutants which were gtfS, gtfS/gtfT, gtfS/gtfI and gtfI. The mutants were tested for their ability to accumulate on wires and on plastic microtiter trays in the presence of sucrose. The gtfS mutant displayed a reduced ability to adhere compared to the wild type but there was no further reduction of adherence in a gtfS/gtfT mutant. In contrast, the gtfS/gtfI double mutant showed a drastic reduction in adherence and when gtfI alone was inactivated, bacteria were unable to adhere to a hard surface. The results confirmed that insoluble glucan is required for strong adherence to a smooth surface but that the amount and structure of this glucan is dependent upon the availability of soluble glucans to act as primer molecules.

Molecular approaches to the identification of streptococci.

The ability to identify rapidly organisms to the species, and at times subspecies level, is an important step in the treatment of bacterial infections and for monitoring the spread of microorganisms. Conventional identification of streptococci relies on the isolation and culturing of bacterial cells, and then submitting the culture to a battery of biochemical tests. Whereas these panels are useful and have a fairly high degree of accuracy, they can suffer from preparation time and problems with the identification of nutritionally variant strains (mainly with the viridans streptococci). Biochemical classification also lacks the ability to type species to the level of a particular clonal population or strain. Although not as important from the diagnostic perspective, the ability to type bacteria to the clonal level is important for epidemiologic studies of disease outbreaks.

The multiple-sugar metabolism (msm) gene cluster of Streptococcus mutans is transcribed as a single operon.

The multiple-sugar metabolism (msm) locus of Streptococcus mutans constitutes a non-PTS sugar uptake system responsible for the transport and utilization of raffinose, melibiose and isomaltotrioses. While previous studies have used polar mutations to suggest that these genes are co-transcribed, there has not been evidence to support this. In this report we present direct evidence that the msm genes can be transcribed as a single operon.

Femoral neck fractures in the elderly patient: a preventable injury.

There is growing evidence that many fractures of the femoral neck in the elderly occur spontaneously because of stresses imposed on osteoporotic bone, rather than because of the trauma of the fall. A case of a spontaneous femoral neck fracture (Garden type IV) in an elderly woman with osteoporosis is presented. Early detection and medical management of osteoporosis complemented with assistive technology could have prevented this common injury.

Demineralized bone matrix in the stabilization of porous-coated implants in bone defects in rabbits.

Three types of grafts were investigated in rabbits to measure fixation strength of bony ingrowth into porous-coated titanium alloy implants. Autogeneic iliac crest bone (ABG), allogeneic demineralized bone matrix (DBM), and DBM augmented with fibrin glue (DBM + FG) grafts were compared with a press-fit implant control group. Initially, the ABG group required eight weeks and the DBM and DBM + FG groups 12 weeks to achieve fixation strength similar to that of the press-fit group at four weeks. Strength increased with time and at 16 weeks reached 83 kg in the ABG group, 71 kg in the DBM group, and 79 kg in the DBM + FG group, compared with 86 kg for the press-fit group. The ratio of the DBM and the DBM + FG group push-out forces to the ABG push-out force improved from 40% at four weeks to more than 80% at 16 weeks. Histologic analysis of bone ingrowth showed that at 12 weeks, bony ingrowth accounted for 21%, 22%, 16%, and 32% of the porous area in the ABG, DBM, DBM + FG, and press-fit groups, respectively. After eight weeks, there was no statistical difference between DBM, DBM + FG, and ABG grafts in either strength or bony ingrowth. The results demonstrate that over long periods, DBM grafts provide fixation stability comparable with that of autogeneic bone graft.

Transfibular compression arthrodesis of the ankle joint.

Eleven ankle arthrodeses were performed on ten patients using a transfibular technique. This technique employs a lateral approach in which the distal fibula is resected while preserving the peroneal tendon sheath, allowing excellent exposure and contouring of the tibiotalar joint. Compression is attained by using a T-plate placed across the tibiotalar joint with the aid of an AO compression apparatus. At 2.5 years after operation, fusion was achieved in ten ankles. Average time in immobilization was 11.8 weeks, and the fused ankle was cosmetically pleasing. Fusion occurred in nine of 11 ankles fused using other methods during this period. Pin tract infection occurred in three, the time in immobilization averaged 16.2 weeks, and the cosmetic result was inferior.