Exploiting the aggregation propensity of beta-lactamases to design inhibitors that induce enzyme misfolding.

There is an arms race between beta-lactam antibiotics development and co-evolving beta-lactamases, which provide resistance by breaking down beta-lactam rings. We have observed that certain beta-lactamases tend to aggregate, which persists throughout their evolution under the selective pressure of antibiotics on their active sites. Interestingly, we find that existing beta-lactamase active site inhibitors can act as molecular chaperones, promoting the proper folding of these resistance factors. Therefore, we have created Pept-Ins, synthetic peptides designed to exploit the structural weaknesses of beta-lactamases by causing them to misfold into intracellular inclusion bodies. This approach restores sensitivity to a wide range of beta-lactam antibiotics in resistant clinical isolates, including those with Extended Spectrum variants that pose significant challenges in medical practice. Our findings suggest that targeted aggregation of resistance factors could offer a strategy for identifying molecules that aid in addressing the global antibiotic resistance crisis.

Cervicofacial actinomycosis following third molar removal: case-series and review.

Actinomycosis is an opportunistic infection caused by bacteria of the Actinomyces spp., commonly A. israelii. These are non-pathogenic commensals in the mouth, gut, and female genital tract. An infection may arise following trauma or surgery, such as tooth extraction. More than half of cases of actinomycosis occur in the perimandibular area and are termed cervicofacial actinomycosis. Initially, the infection develops as a painful, rapidly progressive swelling. The lesion may then indurate and is often painless while the overlying skin discolors red to purple-blue. Prolonged treatment with antibiotics and surgery are often required for resolution, unless treatment is promptly started. However, diagnosis may be delayed or missed because of difficult bacterial culturing and frequent confusion with malignancy and other infections. This case study describes six patients who developed cervicofacial actinomycosis following third molar extraction. The purpose of this study is to inform clinicians on this stubborn and deceitful disease entity and to highlight the importance of clinical recognition for quick resolution with minimal morbidity.

Efficacy and safety of a Belgian tertiary care outpatient parenteral antimicrobial therapy (OPAT) program.

PURPOSE: Evidence supports the implementation of outpatient parenteral antimicrobial therapy (OPAT) as standard of care. Until 2015 the overall experience with OPAT in Belgium remained limited. The aim of this study was to evaluate the efficacy and safety of a Belgian 'OPAT at home' program, which was implemented in University Hospitals Leuven starting from January 2017. METHODS: A mono-centric, prospective, observational study was carried out. All OPAT cases discharged between 10 January 2017 and 10 January 2019 were included in the study. Relevant demographic and clinical patient data were collected. The outcomes were clinical cure rate, OPAT related readmission rate, adverse event rate and patients' satisfaction. RESULTS: Over the two-year study period, 152 OPAT episodes were started in 130 patients, resulting in 3153 avoided hospitalization days which corresponds to 5.4 freed hospital beds. Urinary tract infections accounted for 40.8% of OPAT courses and temocillin was the most frequently used antibiotic (24.3%). Cure was achieved in 97.9% of the OPAT episodes. During 22 (14.5%) OPAT episodes, patients experienced adverse events, including line related adverse events (7.9%) and adverse drug events (6.6%). An OPAT related readmission rate of 9.2% was observed, mostly related to line-associated adverse events. All patients who completed the satisfaction survey (n = 23) were very satisfied with their OPAT course. CONCLUSION: The University Hospitals Leuven OPAT program is associated with a high level of clinical cure and low all-cause readmission and adverse event rates. Improvement actions are described to further reduce the readmission rate to less than 5.0%.

Translating Data From an Electronic Prescribing and Medicines Administration System Into Knowledge: Application to Doctor-Nurse Time Discrepancy in Antibiotic Ordering and Administration.

BACKGROUND: Electronic Prescribing and Medicines Administration (EPMA) systems are being widely implemented to facilitate medication safety improvement. However, translating the resulting big data into actionable knowledge has received relatively little attention. OBJECTIVE: The objective of this study was to use routinely collected EPMA data in the study of exact time discrepancy between physicians' order and nurses' administration of systemic antibiotics. We evaluated first and follow-up dose administration and dose intervals and examined multifactorial determinants in ordering and administration explaining potential discrepancy. METHODS: We conducted an observational study of electronic health records for all medical patient stays with antibiotic treatment from January to June 2018 (n=4392) in a large Belgian tertiary care hospital. Using an EPMA system with Barcode Medication Administration, we calculated time discrepancy between order and administration of first doses (n=6233), follow-up doses (n=87 960), and dose intervals. Multiple logistic regression analysis estimated the association between time discrepancy and various determinants in ordering and administration. RESULTS: Time discrepancy between physician order and nurse administration was <30 minutes for 48.7% of first doses and 61.7% of follow-up doses, with large variation across primary diagnoses. Greater dose intervals, oral versus intravenous administration, and order diversion from regular nurse administration rounds showed strongest association with less timely administration. CONCLUSIONS: EPMA systems show huge potential to generate actionable knowledge. Concerning antibiotic treatment, having physicians' orders coincide with regular nurse administration rounds whenever clinically appropriate, further taking contextual factors into account, could potentially improve antibiotic administration timeliness.

Supervised teaching and feedback improve physiotherapists' reporting of the International Classification of Functioning, Disability and Health in physiotherapeutic electronic patient records: A proof-of-concept randomized controlled trial.

RATIONALE, AIMS, AND OBJECTIVES: The International Classification of Functioning, Disability and Health (ICF) is a landmark for physiotherapy to describe the full spectrum of human functioning, but ICF patient record completion could improve. In this study, we examine the effect of supervised teaching and personalized feedback on physiotherapists' completion and reporting of ICF in electronic patient records. METHOD: In this proof-of-concept randomized controlled trial, the intervention group (10 physiotherapists) received supervised teaching and four rounds of personalized feedback on reporting of ICF components in electronic patient records. In the intervention group, review on patient record completion (n = 670 records) was performed at baseline, after teaching, after each of four feedback rounds, and at long-term follow-up. In the control group (five physiotherapists), which received no supervised teaching nor personalized feedback, review (n = 140 records) was performed at baseline, after the third feedback round of the intervention group, and at follow-up. RESULTS: After the third round of feedback (95% vs 72% completion; ß, 2.68; 95% CI, 0.62-4.74), patient record completion was significantly higher in the intervention group. This was also true for following ICF components: "activity" (93% versus 64% completion; ß, 3.03; 95% CI, 1.52-4.54), "participation" (50% versus 14% completion; ß, 3.67; 95% CI, 1.79-5.55), and "personal factors" (35% versus 20% completion; ß, 2.10; 95% CI, 0.63-3.57). These statistically significant and clinically relevant effects persisted at long-term follow-up. For "environmental factors," effects after the third round of feedback (75% vs 30% completion; ß, 1.88; 95% CI, 0.63-3.13) disappeared at follow-up. Reporting of "body functions and structures" improved similarly across groups. CONCLUSIONS: Supervised teaching and personalized feedback are active ingredients of an intervention to improve reporting of ICF components in physiotherapeutic patient records.

Outpatient parenteral antimicrobial therapy and antibiotic stewardship: opponents or teammates?

PURPOSE: This narrative review aims to describe barriers of outpatient parenteral antimicrobial therapy at home (OPAT), potentially compromising general standards of antibiotic stewardship (ABS) and facilitators of OPAT for ABS. METHODS: After a literature review, five authors determined the barriers and facilitators to discuss in this review. RESULTS: Sixty-six publications were included in the narrative review and seven barriers and five facilitators are discussed in this article. The impracticability of multiple daily dosing during OPAT, the impact of real-life temperature variations, deviations of the infusion rates of elastomeric devices, access to prolonged intravenous antibiotic therapy, not administering loading doses before the initiation of extended or continuous infusions and the transmural nature of care associated with OPAT, can lead to deviations of recommended treatment regimens and sub-optimal clinical and laboratory follow-up, with a risk of inferior clinical outcomes, adverse events, drug-resistance and higher costs. On the other hand, OPAT provides access to treatments with intravenous antibiotics and simultaneously avoids prolonged hospitalization. CONCLUSION: Implementing ABS guidelines in OPAT programs, e.g., by using a multidisciplinary team approach and facility-specific protocols for OPAT with patient selection criteria and instructions for selection, storage, preparation and administration of antibiotics, can improve appropriate antibiotic use. Additionally, further research should examine the effectiveness of these interventions on outcomes of OPAT.

Compliance with evidence-based guidelines for the prevention of central line-associated bloodstream infections in a Belgian home care setting: An observational study.

This study assessed the compliance of Belgian home care nurses with good practice recommendations to prevent central line-associated bloodstream infections. The compliance to 3 care bundles was 0% (0 out of 7), 13.3% (2 out of 15), and 22.2% (2 out of 9), respectively. This finding is important given the increasing number of home care patients with an intravascular catheter and underscores the need for quality improvement strategies to prevent central line-associated bloodstream infections in home care.

Frequency and Pattern of IgE-mediated Sensitization to Aero and Food Allergens in Ahvaz, Province of Khuzestan in Southwestern Iran.

Efficient diagnosis of allergy and proper treatment need identification of the causative allergens eliciting clinical symptoms. The present study was performed to identify the most common aero- and food allergens and determine the pattern of sensitization among people of Ahvaz (southwestern Iran), one of the most polluted cities worldwide. Based on the physical examination and medical records, patients were referred to the Allergy laboratory for "in vitro" IgE determination. Specific and total IgE was determined by the ImmunoCAP system (Thermo Fisher-Phadia, Uppsala, Sweden). A total of 666 consecutive patients (51.1% female) were tested for 202 different allergens. The majority of requests (57%) belonged to food allergens. Sensitization to at least one allergen was found in 47.6% of patients. In a selected group of allergens for which specific IgE had been tested in at least 100 patients, the most common sensitizing aeroallergens were Russian thistle, grass pollen, and willow; while wheat, honey, and shrimp were the most frequent food allergens, respectively. Sensitization profiles based on measurement of specific IgE indicated that Russian thistle, grasses, and wheat were the most prevalent allergens in people with allergic symptoms living in Ahvaz.

Ses proteins as possible targets for vaccine development against Staphylococcus epidermidis infections.

OBJECTIVES: The opportunistic pathogen Staphylococcus epidermidis is progressively involved in device-related infections. Since these infections involve biofilm formation, antibiotics are not effective. Conversely, a vaccine can be advantageous to prevent these infections. In view of vaccine development, predicted surface proteins were evaluated on their potential as a vaccine target. METHODS: Immunoglobulins directed against S. epidermidis surface proteins SesB, M, O, Q and R were used to firstly affirm their surface location. Further, inhibitory effects of these IgGs on biofilm formation were determined in vitro on polystyrene and polyurethane surfaces and in vivo using a subcutaneous catheter mouse model. We also examined the opsonophagocytotic capacity of these IgGs. RESULTS: Surface localization of the five Ses proteins was demonstrated both for planktonic and sessile cells, though to a variable extent. Ses-specific IgGs added to planktonic cells had a variable inhibitory effect on cell adhesion to polystyrene, while only anti-SesO IgGs decreased cell attachment to polyurethane catheters. Although phagocytic killing was only obtained after opsonization with SesB-specific IgGs, a significant reduction of in vivo formed biofilms was observed after administration of SesB-, SesM- and SesO-specific IgGs. CONCLUSIONS: Regardless of their characterization or function, S. epidermidis surface proteins can be adequate targets for vaccine development aiming the prevention of device-related infections caused by invasive S. epidermidis strains.

Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis.

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichia coli and Acinetobacter baumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy.

Validation of the Child HCAHPS survey to measure pediatric inpatient experience of care in Flanders.

The recently developed Child HCAHPS provides a standard to measure US hospitals' performance on pediatric inpatient experiences of care. We field-tested Child HCAHPS in Belgium to instigate international comparison. In the development stage, forward/backward translation was conducted and patients assessed content validity index as excellent. The draft Flemish Child HCAHPS included 63 items: 38 items for five topics hypothesized to be similar to those proposed in the US (communication with parent, communication with child, attention to safety and comfort, hospital environment, and global rating), 10 screeners, a 14-item demographic and descriptive section, and one open-ended item. A 6-week pilot test was subsequently performed in three pediatric wards (general ward, hematology and oncology ward, infant and toddler ward) at a JCI-accredited university hospital. An overall response rate of 90.99% (303/333) was achieved and was consistent across wards. Confirmatory factor analysis largely confirmed the configuration of the proposed composites. Composite and single-item measures related well to patients' global rating of the hospital. Interpretation of different patient experiences across types of wards merits further investigation. CONCLUSION: Child HCAHPS provides an opportunity for systematic and cross-national assessment of pediatric inpatient experiences. Sharing and implementing international best practices are the next logical step. What is Known: • Patient experience surveys are increasingly used to reflect on the quality, safety, and centeredness of patient care. • While adult inpatient experience surveys are routinely used across countries around the world, the measurement of pediatric inpatient experiences is a young field of research that is essential to reflect on family-centered care. What is New: • We demonstrate that the US-developed Child HCAHPS provides an opportunity for international benchmarking of pediatric inpatient experiences with care through parents and guardians. • Our study findings show considerable variation in experiences for types of pediatric services. Support to share good practices and launch quality improvement initiatives can be obtained by organizing regular two-way feedback sessions with clinicians to place the findings in context.

De novo design of a biologically active amyloid.

Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional-determined by VEGFR2 loss of function in a biological context in which target protein function is essential.

sesC as a genetic marker for easy identification of Staphylococcus epidermidis from other isolates.

Staphylococcus epidermidis is one of the major concerns with respect to hospital-acquired infections. Therefore, a rapid and easy method to identify at species level S. epidermidis isolates out of a broad range of bacteria is necessary. Based on earlier studies, the sesC gene encoding a S. epidermidis surface protein revealed to be a highly conserved gene in this species. By means of an easy and inexpensive PCR assay, the presence of sesC was checked in 438 clinical staphylococcal isolates. Results showed that sesC is specifically present in all S. epidermidis. In conclusion, the sesC gene can be exploited as a genetic marker in order to distinguish S. epidermidis from other isolates.

The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation.

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325-4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325-4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325-4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325-4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325-4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.

Protein aggregation as an antibiotic design strategy.

Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.

Performance of strip-based glucose meters and cassette-based blood gas analyzer for monitoring glucose levels in a surgical intensive care setting.

BACKGROUND: We verified the analytical performance of strip-based handheld glucose meters (GM) for prescription use, in a comparative split-sample protocol using blood gas samples from a surgical intensive care unit (ICU). METHODS: Freestyle Precision Pro (Abbott), StatStrip Connectivity Meter (Nova), ACCU-CHEK Inform II (Roche) were evaluated for recovery/linearity, imprecision/repeatability. The GMs and the ABL90 (Radiometer) blood gas analyzer (BGA) were tested for relative accuracy vs. the comparator hexokinase glucose-6-phosphate-dehydrogenase (HK/G6PDH) assay on a Cobas c702 analyzer (Roche). RESULTS: Recovery of spiked glucose was linear up to 19.3 mmol/L (347 mg/dL) with a slope of 0.91-0.94 for all GMs. Repeatability estimated by pooling duplicate measurements on samples below (n=9), in (n=51) or above (n=80) the 4.2-5.9 mM (74-106 mg/dL) range were for Freestyle Precision Pro: 4.2%, 4.0%, 3.6%; StatStrip Connectivity Meter: 4.0%, 4.3%, 4.5%; and ACCU-CHEK Inform II: 1.4%, 2.5%, 3.5%. GMs were in agreement with the comparator method. The BGA outperformed the GMs, with a MARD of 3.9% compared to 6.5%, 5.8% and 4.4% for the FreeStyle, StatStrip and ACCU-CHEK, respectively. Zero % of the BGA results deviated more than the FDA 10% criterion as compared to 9.4%, 3.7% and 2.2% for the FreeStyle, StatStrip and ACCU-CHEK, respectively. For all GMs, icodextrin did not interfere. Variation in the putative influence factors hematocrit and O2 tension could not explain observed differences with the comparator method. CONCLUSIONS: GMs quantified blood glucose in whole blood at about the 10% total error criterion, proposed by the FDA for prescription use.

Selectivity of aggregation-determining interactions.

Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein ß-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichia coli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.

Novel LC-MS/MS method for plasma vancomycin: comparison with immunoassays and clinical impact.

BACKGROUND: Accurate quantification of vancomycin in plasma is important for adequate dose-adjustment. As literature suggests between-method differences, our first objective was to develop a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for total vancomycin in human plasma and to compare frequently used immunoassays with this method. Secondly, we investigated the clinical impact of between-method quantification differences. METHODS: For LC-MS/MS, lithium heparin plasma was extracted by adding a precipitation reagent containing the internal standard (vancomycin-des-leucine). Analysis was performed on an Acquity TQD mass spectrometer equipped with an Acquity UPLC 2795 separations module. Our method was analytically validated and compared with four frequently used immunoassays from four different manufacturers. Vancomycin concentrations were clinically classified as toxic, therapeutic and sub-therapeutic. Clinical discordance was calculated using LC-MS/MS as a reference. RESULTS: A novel LC-MS/MS method using protein precipitation as sole pretreatment and an analysis time of 5.0 min was developed. The assay had a total imprecision of 2.6-8.5%, a limit of quantification of 0.3 mg/L and an accuracy ranging from 101.4 to 111.2%. Using LC-MS/MS as reference, three immunoassays showed a mean proportional difference within 10% and one showed a substantial mean proportional difference of >20%. Clinical discordant interpretation of the obtained concentrations ranged from 6.1 to 22.2%. CONCLUSIONS: We developed a novel LC-MS/MS method for rapid analysis of total vancomycin concentrations in human plasma. Correlation of the method with immunoassays showed a mean proportional difference >20% for one of the assays, causing discordant clinical interpretation in more than 1 out of 5 samples.

Non-typeable Haemophilus influenzae, an under-recognised pathogen.

Non-typeable Haemophilus influenzae (NTHi) is a major cause of mucosal infections such as otitis media, sinusitis, conjunctivitis, and exacerbations of chronic obstructive pulmonary disease. In some regions, a strong causal relation links this pathogen with infections of the lower respiratory tract. In the past 20 years, a steady but constant increase has occurred in invasive NTHi worldwide, with perinatal infants, young children, and elderly people most at risk. Individuals with underlying comorbidities are most susceptible and infection is associated with high mortality. ß-lactamase production is the predominant mechanism of resistance. However, the emergence and spread of ß-lactamase-negative ampicillin-resistant strains in many regions of the world is of substantial concern, potentially necessitating changes to antibiotic treatment guidelines for community-acquired infections of the upper and lower respiratory tract and potentially increasing morbidity associated with invasive NTHi infections. Standardised surveillance protocols and typing methodologies to monitor this emerging pathogen should be implemented. International scientific organisations need to raise the profile of NTHi and to document the pathobiology of this microbe.