Malleus

Complete, draft genomes, and raw reads of Burkholderia spp. including former Burkholderia genomes from the newly renamed genera Paraburkholderia and Caballeronia(Table S3) were analyzed using PhaME. Genomes from genera that have multiple available genomes were randomly selected to have a mixture of complete and draft genomes. Ralstonia solanacearum PSI07 was also included and used as an outgroup and PhaME picked B. mallei NCTC 10247 as the reference genome based on MinHash distances. Raw reads were first quality controlled using FaQCs v2.09^{81 (link)} and then added to PhaME analysis. Orthologous polymorphic positions were kept and used to build a maximum likelihood tree using RAxML (GTRGAMMAI) with 100 bootstrap supports.
Subsets of the genomes that belong to the Bcc or the B. pseudomallei groups were further analyzed using PhaME (Table S3). Genomes that belong to the corresponding clades were selected from the whole Burkholderia tree and the original alignments were used to recalculate the core genome and core SNPs, which were then used to reconstruct maximum likelihood tree using RAxML (GTRGAMMAI) with 100 bootstraps.

Whole-cell (WC) antigen was extracted from the wild type strain B. pseudomallei K96243 (from a Thai patient in northeast Thailand; expresses type A OPS) and an OPS mutant (ΔwbiD K96243) by heating at 80°C for 1 h. The supernatant was used as the antigen described previously [21 (link), 26 (link)]. The OPS mutant defective in wbiD (BPSL2677) was constructed as described in our previous study [27 (link)].
B. pseudomallei LPS type A was extracted from the select agent excluded strain RR2808 (capsule mutant) using a modified hot phenol method [28 (link), 29 (link)]. Purified OPS antigen was then obtained using acid hydrolysis and gel permeation chromatography as previously described [30 (link)]. For expression of recombinant Hcp1 (rHcp1) with a N-terminal 6xHis-Tag, the hcp1 ORF (BMAA0742) was PCR amplified from B. mallei ATCC 23344 genomic DNA using the Bmhcp1-6HisF (5’-CCCAACGGTCTCACATGGCGGCGCATCATCATCATCATCATCTGGCCGGAATATATCTCAAGG-3’) and Bmhcp1-R1 (5’-CCCAACGGTCTCAAGCTTCAGCCATTCGTCCAGTTTGCGGC-3’) primer pair; BsaI linkers are underlined. The resulting DNA fragment was digested with BsaI and cloned into pBAD/HisA digested with NcoI/HindIII producing plasmid pBADBmhcp1-6HisF. Notably, B. pseudomallei and B. mallei Hcp1 proteins are 99.4% identical. Recombinant DNA techniques were conducted as previously described [31 (link)]. Oligonucleotide primers were obtained from Integrated DNA Technologies. DNA sequencing was performed by ACGT Inc. For purification of rHcp1, E. coli TOP10 (pBADBmhcp1-6HisF) was grown to mid log phase in LB broth and protein expression was induced using 0.02% L-arabinose (Sigma). Bacterial pellets were resuspended in B-PER (Pierce) plus Benzonase (Novagen) and Lysozyme (100 μg/ml) and incubated for 20 min at room temperature with gentle agitation. Insoluble material was removed by centrifugation and the resulting supernatant was loaded onto a gravity-fed Ni-NTA agarose (Invitrogen) column. The column was washed with Wash Buffer (50 mM Tris pH 8.0, 300 mM NaCl and 40 mM Imidazole), protein was eluted with Elution Buffer (50 mM Tris pH 8.0, 50 mM NaCl and 300 mM Imidazole) then dialyzed against PBS and loaded onto a gravity-fed His-Pur Cobalt Resin (Thermo Scientific) column. The column was washed with PBS and rHcp1 was eluted with Wash Buffer, dialyzed against PBS, concentrated and stored at 4°C. Protein concentrations were determined using a BCA protein assay kit (Pierce). Endotoxin removal was performed using High Capacity Endotoxin Removal Resin (Pierce). The amount of endotoxin in the rHcp1 preparations was quantitated using a LAL Chromogenic Endotoxin Quantitation Kit (Pierce).

Intraoperative images of tympanic membranes were collected from pediatric patients undergoing myringotomy with possible tympanostomy tube placement for recurrent acute otitis media or otitis media with effusion between November 2019 and September 2020. Consent was exempted under the IRB listed above. Inclusion criteria for the images were greater than 75% visibility of the tympanic membrane, sufficient image quality for distinguishing major anatomic landmarks (i.e., annular ligament, malleus umbo), and normal appearance of the tympanic membrane without middle ear fluid as ascertained by myringotomy. Images were taken using a 0-degree 2.7 mm Hopkins rod telescope coupled to a high-definition (HD) camera (Karl Storz SE & Co KG, Tuttlingen, Germany), which captured images at 1920 x 1080 pixel resolution.

The study was conducted in accordance with the principles of the Helsinki Declaration and was approved by the internal Ethical Committee. Data were examined in agreement with the Italian privacy and sensible data laws. Consecutive COM patients treated with canal wall up tympanoplasty at Otorhinolaryngology Unit of Vicenza Civil Hospital (Italy) were enrolled in this retrospective study. The inclusion criteria were the following: (i) COM with TM perforation; (ii) mastoidectomy performed together with tympanoplasty; (iii) age > 18 years old. Patients with craniofacial dysmorphisms, cholesteatoma, ossiculoplasty, or revision surgery were excluded.
Demographic and clinical characteristics were recorded. At first and follow-up visits, all patients underwent otomicroscopy and pure tone audiometry in a silent cabin, including bone-conduction (BC) thresholds at 0.5, 1, 2, and 4 kHz, and air-conduction (AC) thresholds at 0.25, 0.5, 1, 2, 4, and 8 kHz, for both ears (operated and contralateral) [10 (link)]. When necessary, in order to eliminate the interaction of the contralateral ear caused by interaural attenuation, a narrow band noise was administered to the better ear while testing the worst [11 (link)]. Air-bone gap (ABG) was calculated as the average difference between the air- and bone-conduction thresholds at 0.5, 1, 2, and 4 kHz. Only AC and BC results that were obtained at the same time were used for calculations, according to American Academy of Otolaryngology Head and Neck Surgery guidelines [12 (link)]. TM perforation was classified according to size (one, two, three, or all quadrants), as previously reported [13 (link)]. Otorrhea was classified according to Bellucci [14 (link)] as (i) dry; (ii) occasionally wet; or (iii) persistently wet.
Tympanoplasty and concurrent mastoidectomy was performed with a postauricular approach using the microscope in all patients. A knife was used to scratch the edges of the perforation circumferentially. The tympanomeatal flap was elevated. The tympanic cavity was visualized, and ossicles condition was surveyed. If pathologic lesions were detected (e.g., tympanosclerotic lesions, granulation, or fibrosis), they were removed to mobilize the ossicles. We excluded the cases with poor ossicle mobility in which ossiculoplasty was a better indication. The TM perforation was closed with a medial-to-malleus underlay technique. Different graft types were used: allograft pericardium (Tutoplast; ENTrigue Biologics, San Antonio, TX, USA), xenograft (porcine submucosal collagen; Biodesign; Cook Medical Inc., Bloomington, IN, USA), and autografts (dry temporalis fascia or tragal cartilage). Four surgeons operated on the patients, and every surgeon chose the graft type independently, according to their own evaluation and preference. Graft size was two to three times larger than TM perforation. Intraoperative bleeding was classified as low, moderate, or severe. Intraoperative complications were recorded.
The general follow-up schedule (adjustable to patient’s individual characteristics) was as follows: (i) every 15 days in the first month after surgery; (ii) once every month in the second and third month; (iii) every 6 months thereafter. We recorded postoperative complications. Postoperative infection was defined as an infection resulting in a patient being prescribed an antibiotic during the first month of follow-up. For postoperative ABG, we considered the value at last follow-up visit.
As outcome variable, we considered postoperative perforation. In postoperative TM perforation, we included persistent perforation (a perforation that did not heal within 3 months after surgery) and recurrent perforation (patients who had documented closure and a subsequent perforation were identified).