Mupirocin

The de novo-assembled genomes were interrogated with BLAST+ (v 2.2.28+) blastn and tblastn (9 (link)) to identify nucleotide sequences matching genes from the panel and their matching protein sequences, respectively. The parameters for the two programs were as follows: for blastn, word size of 17, gap opening penalty of 5, and gap extension penalty of 2; for tblastn, word size of 3, gap opening penalty of 11, and gap extension penalty of 1. The E-value cutoff was set at 0.001. Relative coverage was defined as the product of the proportion of reference allele matched and the sequence identity of the match. For the initial algorithm (v1.0), a relative coverage threshold of >80% was chosen to define gene presence with a high degree of similarity to the reference, based on pilot data (for example, 95% relative coverage may be 95% of the gene length with 100% identity or 100% of the gene length with 95% identity). For housekeeping genes, where resistance is conferred by one or more point mutations, differences between the tblastn result and the query protein sequence were compared to the sequence of the wild-type protein and compared against the catalogue of known antimicrobial resistance-encoding mutations compiled above. Changes in protein sequence (at the same or different codons) which were not previously reported as conferring resistance were counted as susceptible.
To determine the diversity of the isolates tested, in silico prediction of multilocus sequence type (MLST) was also performed using BLAST+. The S. aureus MLST alleles were extracted from assemblies based on sequence similarity to allele 1 for each locus, and the online MLST database (http://saureus.mlst.net/) was used to predict the ST.
The initial development of the algorithm was not done in a blind manner, using 501 clinical S. aureus isolates which had been sequenced and phenotyped previously and whose WGS and resistance data were available (“derivation set”). To ensure a representative range of sequence types, isolates were identified from bacteremia and carriage collections held at the Oxford Radcliffe Hospitals NHS Trust and Brighton and Sussex University Hospitals NHS Trust, spanning a period of 13 years (Table 3) (10 (link)). The collection included 159 MRSA isolates (32%). All isolates had been tested at each site by the routine clinical laboratories for resistance to a standard first-line panel of antimicrobial agents (penicillin, methicillin, erythromycin, vancomycin, ciprofloxacin, tetracycline, gentamicin, fusidic acid, and rifampin at both sites; mupirocin and clindamycin for Brighton isolates only; trimethoprim for Oxford isolates only). In the Brighton clinical laboratory, susceptibility testing was performed using the Vitek automated system (bioMérieux, Basingstoke, United Kingdom), and in the Oxford clinical laboratory, isolates were phenotyped by disc diffusion (11 (link)). The susceptibility testing results were retrieved electronically from laboratory databases. Methicillin resistance was tested using cefoxitin (Brighton) or oxacillin (Oxford).

We cultured MRSA from screening swabs and clinical specimens by plating on to MRSA selective medium (Brilliance MRSA chromogenic medium, Oxoid, Basingstoke, UK). We identified bacteria with a commercial latex agglutination kit (Pastorex Staph Plus, Bio Rad Laboratories, Hemel Hempstead, UK). We did antimicrobial susceptibility testing with disk diffusion¹² for the following drugs: cefoxitin, ciprofloxacin, erythromycin, fusidic acid, gentamicin, mupirocin, neomycin, rifampicin, tetracycline, and vancomycin. All isolates were assigned unique strain identification numbers. We prepared sequencing libraries from 500 ng of DNA extracted from each MRSA isolate as previously described,¹³ with amplification using Kapa Hifi polymerase (Kapa Biosystems, Woburn, MA, USA). Whole-genome sequencing was done with an Illumina MiSeq (Illumina, San Diego, CA, USA) to generate 150 bp paired end reads, and interpreted by an investigator (SRH) who was masked to all clinical, epidemiological, and microbiological information. The genome data has been deposited in the European Nucleotide Archive (appendix). We aligned sequence reads to the chromosome (accession number HE681097) and plasmid (CP002148) of a reference isolate (HO 5096 0412) to identify single-nucleotide polymorphisms (SNPs) and insertions or deletions. This reference isolate was defined by multilocus sequence typing as sequence type (ST) 22.

A tongue-shaped flap was created on the radial wall of the 5th digit, with the longitudinal edges not exceeding the radial surface of the digit, and the distal edge made slightly beyond the PIP joint line. To ensure full coverage of the volar skin defect, the flap was made 2 mm larger in diameter than the recipient site (Fig. 2).Figure 2

Representative illustrations of camptodactyly of the 5th digit. (a) Frontal and lateral views of the 5th digit before surgery. (b) Design of the tongue-shaped flap, with the longitudinal edges limited within the radial surface, and the distal edge made slightly exceeding the proximal interphalangeal joint line. (c) The volar incision. (d) The lateral view of the digit flap transfer, with direct suturing performed for closure of the donor site. (e) The volar view of the digit after flap transfer, with complete coverage of the volar skin defect.

While creating the edges of the flap, care was taken to preserve the perforating blood vessels of the proper palmar digital arteries, as well as the proper palmar digital nerves.
Sequential release of affected soft tissues was performed in the following order—skin, subcutaneous fibrous fascia, flexor digitorum superficialis tendon, lumbrical muscle insertions if present, and volar plate. The degree of passive extension of the PIP joint was repeatedly tested, and surgical release was considered complete upon achieving full passive extension of the joint. Kirschner (K)-wire fixation was performed following volar plate release.
The radial flap was rotated 90° to cover the volar skin defect, and direct suturing was performed to close the donor site. Free skin grafting was indicated in the presence of high suture tension.
Mupirocin ointment and petroleum jelly (Vaseline) were subsequently applied, and the wound was wrapped with clean dressing. All digits were immobilized in the extended position with a cast for three weeks.

For each combination of microorganism, antimicrobial and food category/animal population were tested, MIC distributions were tabulated in frequency tables, giving the number of isolates tested that have a given MIC at each test dilution (mg/L) of the antimicrobial. Isolate‐based dilution results allowed MIC distributions reported:

for Salmonella for amikacin, ampicillin, azithromycin, cefepime, cefotaxime, cefotaxime and clavulanic acid, ceftazidime, ceftazidime and clavulanic acid, cefoxitin, chloramphenicol, ciprofloxacin, colistin, ertapenem, gentamicin, imipenem, meropenem, nalidixic acid, sulfamethoxazole, temocillin, tetracycline, tigecycline and trimethoprim;

for Campylobacter for chloramphenicol, ciprofloxacin, ertapenem, erythromycin, gentamicin, nalidixic acid and tetracycline;

for indicator E. coli for amikacin, ampicillin, azithromycin, cefepime, cefotaxime, cefotaxime and clavulanic acid, ceftazidime, ceftazidime and clavulanic acid, cefoxitin, chloramphenicol, ciprofloxacin, colistin, ertapenem, gentamicin, imipenem, meropenem, nalidixic acid, sulfamethoxazole, temocillin, tetracycline, tigecycline and trimethoprim;

for MRSA for cefoxitin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, fusidic acid, gentamicin, kanamycin, linezolid, mupirocin, penicillin, quinupristin/dalfopristin, rifampicin, streptomycin, sulfamethoxazole, tetracycline, tiamulin, trimethoprim and vancomycin.

All the patients received standard ICU supportive care and specific COVID-19 therapies according to WHO guidelines [14 ] and national protocols [15 (link)] for treating COVID-19 patients. In addition, the local protocol allowed the use of methylprednisolone at 2 mg/kg/day to prevent the onset of pulmonary fibrosis in patients with acute respiratory distress syndrome (ARDS) who maintained at a PaO₂/FiO₂ ratio < 150 mmHg for at least seven days of MV [16 (link)] and Tocilizumab (TOCI) in patients with moderate or severe ARDS since March 2020. The internal protocol for VAP prevention in use before the pandemic (supplemental material) was routinely applied in all COVID-19 patients with invasive MV. The protocol also included using nasal mupirocin for five days and chlorhexidine for body cleaning at least once daily. However, the high incidence of VAP observed in the first six months of the pandemic (February–April and September–November 2020) led to an urgent multifaceted program (including audit and educational meetings (by electronic platforms) and practical simulation) for reinforcing compliance with the internal protocol. Due to the persistently high rate of VAP observed in the three months following the multifaceted program, from the end of April 2021, we decided to also introduce SDD into the protocol. The SDD consisted of a tobramycin sulfate, colistin sulfate, and amphotericin B suspension that was applied in the patient’s oropharynx and the stomach via a nasogastric tube four times per day for ten consecutive days or until endotracheal tube removal.