Isolation and characterization of a VHH targeting the Acinetobacter baumannii cell surface protein CsuA/B.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A. baumannii outer membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from four A. baumannii strains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intact A. baumannii cells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surface A. baumannii targets could provide tools for further study and treatment of this pathogen. KEY POINTS: •Llama immunization with bacterial OMV preparations for VHH generation •A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target •High-affinity and specific VHH binding to CsuA/B and A. baumannii cells.

Epitope mapping of a blood-brain barrier crossing antibody targeting the cysteine-rich region of IGF1R using hydrogen-exchange mass spectrometry enabled by electrochemical reduction.

Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood-brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen-deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics.

The development and testing of a co-designed distance learning programme to improve the knowledge of trained auxiliary nurse midwives in normal midwifery practice in central India.

BACKGROUND: In the Indian State of Madhya Pradesh maternal health is poor, and women's social status is low. For some women, autonomy and decision-making within the family is limited as they seek medical treatment through their husband or father-in-law. The then state government identified a need to strengthen midwifery care given by auxiliary nurse midwives to improve maternal and neonatal health.>. METHODS: This cross-cultural, two phase study was designed in partnership with an Indian non-governmental organisation, utilising Elliot's action research model within the paradigm of critical theory. Phase 1 investigated the then current situation and established a potential solution to strengthening midwifery practice within Madhya Pradesh. This comprised an educational approach using a specifically designed self-directed distance learning programme focussing on normal pregnancy and childbirth. The distance learning programme was a hard copy workbook supported by a multimedia resource informed by the Rowntree and Analysis Design Development Implementation Evaluation models of educational material development. Phase 2 involved the use and evaluation of the distance learning programme with an initial primary group sample of 28 comprising auxiliary nurse midwives (22), lady health visitors (4) and staff nurses (2) in Madhya Pradesh. Fourteen auxiliary nurse midwife participants completed knowledge tests using a pre- and post-programme multiple choice question paper. This primary group negotiated a 3-day workshop to cover identified gaps within their original 12-week study period. Nineteen additional auxiliary nurse midwife participants joined this workshop (group 2). RESULTS: The multiple choice question test results indicated that the first group had poor knowledge of the normal process of pregnancy and childbirth. After the workshop, they marginally improved their personal performance scores. The second group demonstrated greater change, which suggests that coupling self-directed guided study material with an enabling, face-to-face environment can be successful. CONCLUSIONS: Distance learning among auxiliary nurse midwives in central India may be enhanced when accompanied by face-to-face workshops. Partnership working - at organisational, team and individual levels, was a crucial, and empowering, component of this cross-cultural action research study. Both points have relevance for others undertaking similar studies.

Ketamine as an Analgesic Adjuvant in Adult Trauma Intensive Care Unit Patients With Rib Fracture.

BACKGROUND: Rib fracture associated pain is difficult to control. There are no published studies that use ketamine as a therapeutic modality to reduce the amount of opioid to control rib fracture pain. OBJECTIVE: To examine the analgesic effects of adjuvant ketamine on pain scale scores in trauma intensive care unit (ICU) rib fracture. METHODS: This retrospective, case-control cohort chart review evaluated ICU adult patients with a diagnosis of ≥1 rib fracture and an Injury Severity Score >15 during 2016. Patients received standard-of-care pain management with the physician's choice analgesics with or without ketamine as a continuous, fixed, intravenous infusion at 0.1 mg/kg/h. RESULTS: A total of 15 ketamine treatment patients were matched with 15 control standard-of-care patients. Efficacy was measured via Numeric Pain Scale (NPS)/Behavioral Pain Scale (BPS) scores, opioid use, and ICU and hospital length of stay. Safety of ketamine was measured by changes in vital signs, adverse effects, and mortality. Average NPS/BPS, severest NPS/BPS, and opioid use were lower in the ketamine group than in controls (NPS: 4.1 vs 5.8, P < 0.001; severest NPS: 7.0 vs 8.9, P = 0.004; opioid use: 2.5 vs 3.5 mg morphine equivalents/h/d, P = 0.015). No difference was found between the cohort's length of stay or mortality. Average diastolic blood pressure was higher in the treatment group versus the control group (75.3 vs 64.6 mm Hg, P = 0.014). CONCLUSION: Low-dose ketamine appears to be a safe and effective adjuvant option to reduce pain and decrease opioid use in rib fracture.

Genome and proteome of Campylobacter jejuni bacteriophage NCTC 12673.

Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms.

Orally administered P22 phage tailspike protein reduces salmonella colonization in chickens: prospects of a novel therapy against bacterial infections.

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans.

Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation.

Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in developed countries, and yet little is known concerning the mechanisms by which this fastidious organism survives within its environment. We have demonstrated that C. jejuni 11168 can form biofilms on a variety of surfaces. Proteomic analyses of planktonic and biofilm-grown cells demonstrated differences in protein expression profiles between the two growth modes. Proteins involved in the motility complex, including the flagellins (FlaA, FlaB), the filament cap (FliD), the basal body (FlgG, FlgG2), and the chemotactic protein (CheA), all exhibited higher levels of expression in biofilms than found in stationary-phase planktonic cells. Additional proteins with enhanced expression included those involved in the general (GroEL, GroES) and oxidative (Tpx, Ahp) stress responses, two known adhesins (Peb1, FlaC), and proteins involved in biosynthesis, energy generation, and catabolic functions. An aflagellate flhA mutant not only lost the ability to attach to a solid matrix and form a biofilm but could no longer form a pellicle at the air-liquid interface of a liquid culture. Insertional inactivation of genes that affect the flagellar filament (fliA, flaA, flaB, flaG) or the expression of the cell adhesin (flaC) also resulted in a delay in pellicle formation. These findings demonstrate that the flagellar motility complex plays a crucial role in the initial attachment of C. jejuni 11168 to solid surfaces during biofilm formation as well as in the cell-to-cell interactions required for pellicle formation. Continued expression of the motility complex in mature biofilms is unusual and suggests a role for the flagellar apparatus in the biofilm phenotype.

Quality school: an innovative approach to laboratory quality practices training.

The quality management program in the Division of Transfusion Medicine at our institution had evolved to the point that the program generally was perceived to be the sole responsibility of our Quality Unit (which was administratively independent of day-to-day operations). It became clear that this administrative model was counterproductive to our new goal of instilling a responsibility for quality into every work level of our division. Such a culture change requires a considerable, organized educational effort. Quality School was established to meet these particular educational needs. The details of the modular structure of the courses and the initial results of their implementation are described. This Quality School approach was developed specifically for transfusion medicine, but the principles could be applied to any clinical laboratory.

Antenatal care: provision and inequality in rural north India.

The objectives of this paper are to examine factors associated with use of antenatal care in rural areas of north India, to investigate access to specific critical components of care and to study differences in the pattern of services received via health facilities versus home visits. We used the 1998-1999 Indian National Family Health Survey of ever-married women in the reproductive age group and analysed data from the states of Bihar, Madhya Pradesh, Rajasthan, and Uttar Pradesh (n = 11,369). Overall, about three-fifths of rural women did not receive any antenatal check-up during their last pregnancy. Services actually received were predominantly provision of tetanus toxoid vaccination and supply of iron and folic acid tablets. Only about 13% of pregnant women had their blood pressure checked and a blood test done at least once. Women visited by health workers received fewer services compared to women who visited a health facility. Home visits were biased towards households with a better standard of living. There was significant under-utilisation of nurse/midwives in the provision of antenatal services and doctors were often the lead providers. The average number of antenatal visits reported in this study was 2.4 and most visits were in the second trimester. Higher social and economic status was associated with increased chances of receiving an antenatal check-up, and of receiving specific components including blood pressure measurement, a blood test and urine testing but not the obstetric physical examination, which was however linked to ever-use of family planning and the education of women and their husbands. Thus, pregnant women from poor and uneducated backgrounds with at least one child were the least likely to receive antenatal check-ups and services in the four large north Indian states. Basic antenatal care components are effective means to prevent a range of pregnancy complications and reduce maternal mortality. The findings indicate substantial limitations of the health services in overcoming socio-economic and cultural barriers to access.

Ordering blood for the wrong patient--getting inside the minds of ordering physicians.

OBJECTIVE: To assess the impact on ordering errors when physicians stopped handwriting patient identifiers on requests for blood transfusion. MATERIAL AND METHODS: Physicians, frustrated by the amount of time required to complete paper forms to order blood, asked if the requirement for handwritten patient identifiers, which were in addition to such information "stamped" on blood requests, could be eliminated. We acquiesced to the request, modified the blood ordering forms accordingly, and continued to monitor ordering errors. RESULTS: After elimination of the handwritten identifiers in 1997, ordering errors increased from an annual rate of 1 in 10,000 to 6 in 10,000 blood requests by late 1999. We alerted the clinicians by newsletter, and the rate decreased somewhat (3 in 10,000 requests). However, the error rate did not decrease to its previous level of 1 in 10,000 requests until mid-2001, about 2 1/2 years after reinstitution of the requirement for handwritten patient identifiers. CONCLUSION: An obligatory second entry of demographic identifiers on a blood order requires ordering physicians to carefully consider the identity of the patient receiving a transfusion and reduces the likelihood of transfusion of an unintended recipient. Error management tools, such as a predetermined method for planning, reviewing, and documenting all changes, facilitate detection of trends and responses to corrective actions.

Evolution of quality management: integration of quality assurance functions into operations, or "quality is everyone's responsibility".

The management of quality in the Division of Transfusion Medicine at our institution has undergone a lengthy, sometimes painful but always progressive evolution over nearly four decades. Initially, it consisted of one laboratory technologist who was assigned the task of performing certain basic QC checks on a predetermined list of laboratory, collection, and processing steps. This technologist reported directly to the medical director. The tasks gradually grew in volume and complexity so that a four-person quality unit was established, administratively quite separate from the operations and accountable only to the Medical Director. The next stage in the evolutionary process was more revolutionary in scope because it involved a comprehensive cultural shift toward the concept of "quality is everyone's responsibility." The evolutionary process in our institution to date and the planning and organization involved in the direction and management of the evolution itself are described.