The crystal structures of CDD-1, the intrinsic class D ß-lactamase from the pathogenic Gram-positive bacterium Clostridioides difficile, and its complex with cefotaxime.

Class D ß-lactamases, enzymes that degrade ß-lactam antibiotics and are widely spread in Gram-negative bacteria, were for a long time not known in Gram-positive organisms. Recently, these enzymes were identified in various non-pathogenic Bacillus species and subsequently in Clostridioides difficile, a major clinical pathogen associated with high morbidity and mortality rates. Comparison of the BPU-1 enzyme from Bacillus pumilus with the CDD-1 and CDD-2 enzymes from C. difficile demonstrated that the latter enzymes have broadened their substrate profile to efficiently hydrolyze the expanded-spectrum methoxyimino cephalosporins, cefotaxime and ceftriaxone. These two antibiotics are major contributors to the development of C. difficile infection, as they suppress sensitive bacterial microflora in the gut but fail to kill the pathogen which is highly resistant to these drugs. To gain insight into the structural features that contribute to the expansion of the substrate profile of CDD enzymes compared to BPU-1, we solved the crystal structures of CDD-1 and its complex with cefotaxime. Comparison of CDD-1 structures with those of class D enzymes from Gram-negative bacteria showed that in the cefotaxime-CDD-1 complex, the antibiotic is bound in a substantially different mode due to structural differences in the enzymes' active sites. We also found that CDD-1 has a uniquely long Ω-loop when compared to all other class D ß-lactamases. This Ω-loop extension allows it to engage in hydrogen bonding with the acylated cefotaxime, thus providing additional stabilizing interactions with the substrate which could be responsible for the high catalytic activity of the enzyme for expanded-spectrum cephalosporins.

Molecular Basis of Surgical Coaptation Techniques in Peripheral Nerve Injuries.

Peripheral nerve injuries requiring surgical repair affect over 100,000 individuals in the US annually. Three accepted methods of peripheral repair include end-to-end, end-to-side, and side-to-side neurorrhaphy, each with its own set of indications. While it remains important to understand the specific circumstances in which each method is employed, a deeper understanding of the molecular mechanisms underlying the repair can add to the surgeon's decision-making algorithm when considering each technique, as well as help decide nuances in technique such as the need for making epineurial versus perineurial windows, length and dept of the nerve window, and distance from target muscle. In addition, a thorough knowledge of individual factors that are active in a particular repair can help guide research into adjunct therapies. This paper serves to summarize the similarities and divergences of the three commonly used nerve repair strategies and the scope of molecular mechanisms and signal transduction pathways in nerve regeneration as well as to identify the gaps in knowledge that should be addressed if we are to improve clinical outcomes in our patients.

Objective Fluoroscopic Outcomes After Endoscopic Staple Versus Laser Zenker's Diverticulotomy.

OBJECTIVE: Endoscopic Zenker's diverticulotomy (EZD) is typically performed via stapling (endoscopic staple diverticulotomy; ESD) or CO2 laser (endoscopic laser diverticulotomy; ELD). Conflicting reports exist on which approach provides optimal outcomes. This investigation compared objective fluoroscopic data between ESD and ELD. METHODS: A retrospective review of all patients undergoing primary EZD at a tertiary center between January 1, 2014 and January 10, 2022 was performed. Patients undergoing ESD and ELD were matched by preoperative diverticulum size. Primary outcome measures were postoperative diverticulum size and change in diverticulum size from pre- to postoperative swallowing fluoroscopy. Secondary outcome measures were the Eating Assessment Tool (EAT-10) score, penetration aspiration scale (PAS), pharyngeal constriction ratio (PCR), and pharyngoesophageal segment opening (PESo). RESULTS: Thirteen matched pairs with complete fluoroscopic data were identified. The mean (±SD) age of the cohort was 74.0 (±8.5) years. There were no age or gender differences between groups (p > 0.05). The mean pre-operative ZD size was 1.98 (±0.69) cm for ESD and 1.97 (±0.72) cm for ELD; the mean postoperative size was 0.84 (±0.62) cm for ESD and 0.34 (±0.27) cm for ELD (p < 0.05). Mean diverticulum size improved by 1.14 (±0.59) cm after ESD and 1.62 (±0.59) cm after ELD (p < 0.05). There were no significant differences in postoperative EAT-10, PAS, PCR, or PESo between groups. CONCLUSION: The data suggest that endoscopic laser Zenker's diverticulotomy results in a greater improvement in diverticulum size than endoscopic staple diverticulotomy. The data did not suggest a difference in postoperative dysphagia symptom scores or other objective fluoroscopic parameters between staple and laser diverticulotomy. LEVEL OF EVIDENCES: Level 3 Laryngoscope, 133:3057-3060, 2023.

Interventions and Outcomes in Glottic Versus Multi-level Airway Stenosis: A Multi-institutional Review.

OBJECTIVE: Airway stenosis-particularly multi-level-presents complex management challenges. This study assessed rates of tracheostomy, decannulation, and the number of surgeries required in patients with posterior glottic stenosis (PGS), multi-level airway stenosis (MLAS), and bilateral vocal fold paralysis (BVFP). METHODS: Airway stenosis patients treated between 2016 and 2021 at three tertiary medical centers were identified. Demographics, etiology of stenosis, medical comorbidities, and patient-reported outcome measures (PROMs) were collected. RESULTS: 158 patients (84 women, mean age 56.98 ± 15.5 years) were identified (54 PGS, 38 MLAS, and 66 BVFP). 72.3% required tracheostomy, including 72.2%, 86.8%, and 63.6% in these groups, respectively. Decannulation rates were 43.6%, 21.2%, and 32.5% in these groups, respectively. Patients with MLAS had higher rates of tracheostomy than BVFP (p < 0.05). However, decannulation rates were not different between groups (p > 0.05). MLAS required more surgeries (mean 4.0 ± 3.9) than PGS (2.4 ± 2.2, p = 0.02) or BVFP (1.0 ± 1.8, p < 0.0001). Mean PROMs scores at the latest follow-up were abnormal: 15.4 ± 12.2 (Dyspnea Index), 19.9 ± 12.2 (Voice Handicap Index-10), and 9.67 ± 11.1 (Eating Assessment Tool-10). Co-morbidities present included body mass index >30 (41.4%), diabetes (31.8%), pulmonary disease (50.7%), gastroesophageal reflux disease (39.4%), autoimmune disease (22.9%), and tobacco use history (55.2%). CONCLUSIONS: Airway stenosis is a challenging clinical problem that negatively impacts patients' quality of life and often requires numerous surgeries. PGS more frequently requires tracheostomy compared to BVFP, but patients can often decannulate successfully. Patients with multi-level stenosis have lower decannulation rates and require more surgeries than glottic stenosis alone; these patients may benefit from earlier and/or more aggressive intervention. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:528-534, 2023.

Evaluation of a biodegradable polyurethane patch for repair of diaphragmatic hernia in a rat model: A pilot study.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) repair is an area of active research. Large defects requiring patches have a hernia recurrence rate of up to 50%. We designed a biodegradable polyurethane (PU)-based elastic patch that matches the mechanical properties of native diaphragm muscle. We compared the PU patch to a non-biodegradable Gore-Tex™ (polytetrafluoroethylene) patch. METHODS: The biodegradable polyurethane was synthesized from polycaprolactone, hexadiisocyanate and putrescine, and then processed into fibrous PU patches by electrospinning. Rats underwent 4 mm diaphragmatic hernia (DH) creation via laparotomy followed by immediate repair with Gore-Tex™ (n = 6) or PU (n = 6) patches. Six rats underwent sham laparotomy without DH creation/repair. Diaphragm function was evaluated by fluoroscopy at 1 and 4 weeks. At 4 weeks, animals underwent gross inspection for recurrence and histologic evaluation for inflammatory reaction to the patch materials. RESULTS: There were no hernia recurrences in either cohort. Gore-Tex™ had limited diaphragm rise compared to sham at 4 weeks (1.3 mm vs 2.9 mm, p = 0.003), but no difference was found between PU and sham (1.7 mm vs 2.9 mm, p = 0.09). There were no differences between PU and Gore-Tex™ at any time point. Both patches formed an inflammatory capsule, with similar thicknesses between cohorts on the abdominal (Gore-Tex™ 0.07 mm vs. PU 0.13 mm, p = 0.39) and thoracic (Gore-Tex™ 0.3 mm vs. PU 0.6 mm, p = 0.09) sides. CONCLUSION: The biodegradable PU patch allowed for similar diaphragmatic excursion compared to control animals. There were similar inflammatory responses to both patches. Further work is needed to evaluate long-term functional outcomes and further optimize the properties of the novel PU patch in vitro and in vivo. LEVEL OF EVIDENCE: Level II, Prospective Comparative Study.

Laparoscopic repair of bilateral inguinal hernias each containing sigmoid colon in a premature infant.

Inguinal hernias are rare in the general population but are more frequently seen in premature infants. Risk factors include male gender, small for gestational age, low birth weight and respiratory distress. Infant inguinal hernias most frequently contain small bowel. Other contents can include the appendix and cecum, and rarely, the sigmoid colon. Sigmoid colon as content of inguinal hernia in children has only been reported twice in literature, and in both cases it was unilateral. We present the first reported case of bilateral inguinal hernias containing the sigmoid colon in a premature boy, who additionally had the appendix and the cecum in the right hernia. This is also the first reported laparoscopic repair of such a hernia.

Inhibition of the Clostridioides difficile Class D ß-Lactamase CDD-1 by Avibactam.

Avibactam is a potent diazobicyclooctane inhibitor of class A and C ß-lactamases. The inhibitor also exhibits variable activity against some class D enzymes from Gram-negative bacteria; however, its interaction with recently discovered class D ß-lactamases from Gram-positive bacteria has not been studied. Here, we describe microbiological, kinetic, and mass spectrometry studies of the interaction of avibactam with CDD-1, a class D ß-lactamase from the clinically important pathogen Clostridioides difficile, and show that avibactam is a potent irreversible mechanism-based inhibitor of the enzyme. X-ray crystallographic studies at three time-points demonstrate the rapid formation of a stable CDD-1-avibactam acyl-enzyme complex and highlight differences in the anchoring of the inhibitor by class D enzymes from Gram-positive and Gram-negative bacteria.

In Crystallo Time-Resolved Interaction of the Clostridioides difficile CDD-1 enzyme with Avibactam Provides New Insights into the Catalytic Mechanism of Class D ß-lactamases.

Class D ß-lactamases have risen to notoriety due to their wide spread in bacterial pathogens, propensity to inactivate clinically important ß-lactam antibiotics, and ability to withstand inhibition by the majority of classical ß-lactamase inhibitors. Understanding the catalytic mechanism of these enzymes is thus vitally important for the development of novel antibiotics and inhibitors active against infections caused by antibiotic-resistant bacteria. Here we report an in crystallo time-resolved study of the interaction of the class D ß-lactamase CDD-1 from Clostridioides difficile with the diazobicyclooctane inhibitor, avibactam. We show that the catalytic carboxylated lysine, a residue that is essential for both acylation and deacylation of ß-lactams, is sequestered within an internal sealed pocket of the enzyme. Time-resolved snapshots generated in this study allowed us to observe decarboxylation of the lysine and movement of CO2 and water molecules through a transient channel formed between the lysine pocket and the substrate binding site facilitated by rotation of the side chain of a conserved leucine residue. These studies provide novel insights on avibactam binding to CDD-1 and into the catalytic mechanism of class D ß-lactamases in general.