Genomic insights into the expansion of carbapenem-resistant Klebsiella pneumoniae within Portuguese hospitals.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) are a public health concern, causing infections with a high mortality rate, limited therapeutic options and challenging infection control strategies. In Portugal, the CR-KP rate has increased sharply, but the factors associated with this increase are poorly explored. In order to address this question, phylogenetic and resistome analysis were used to compare the draft genomes of 200 CR-KP isolates collected in 2017-2019 from five hospitals in the Lisbon region, Portugal. Most CR-KP belonged to sequence type (ST) 13 (29%), ST17 (15%), ST348 (13%), ST231 (12%) and ST147 (7%). Carbapenem resistance was conferred mostly by the presence of KPC-3 (74%) or OXA-181 (18%), which were associated with IncF/IncN and IncX plasmids, respectively. Almost all isolates were multi-drug resistant, harbouring resistance determinants to aminoglycosides, beta-lactams, trimethoprim, fosfomycin, quinolones and sulphonamides. In addition, 11% of isolates were resistant to colistin. Colonizing and infecting isolates were highly related, and most colonized patients (89%) reported a previous hospitalization. Moreover, among the 171 events of cross-dissemination identified by core genome multi-locus sequence typing data analysis (fewer than five allelic differences), 41 occurred between different hospitals and 130 occurred within the same hospital. The results suggest that CR-KP dissemination in the Lisbon region results from acquisition of carbapenemases in mobile genetic elements, influx of CR-KP into the hospitals by colonized ambulatory patients, and transmission of CR-KP within and between hospitals. Prudent use of carbapenems, patient screening at hospital entry, and improvement of infection control are needed to decrease the burden of CR-KP infection in Portugal.

Antibody immobilization on magnetic particles.

Magnetic particles (MNPs) offer attractive possibilities in biotechnology. MNPs can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide (Fe3O4) MNPs with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat IgG), bovine serum albumin (BSA) as blocking agent, and a complementary antibody labelled with FITC (anti-goat IgG). The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated MNPs, a 5% (w/v) concentration of BSA in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.