Plasmids Shaped the Recent Emergence of the Major Nosocomial Pathogen Enterococcus faecium.

Enterococcus faecium is a gut commensal of humans and animals but is also listed on the WHO global priority list of multidrug-resistant pathogens. Many of its antibiotic resistance traits reside on plasmids and have the potential to be disseminated by horizontal gene transfer. Here, we present the first comprehensive population-wide analysis of the pan-plasmidome of a clinically important bacterium, by whole-genome sequence analysis of 1,644 isolates from hospital, commensal, and animal sources of E. faecium Long-read sequencing on a selection of isolates resulted in the completion of 305 plasmids that exhibited high levels of sequence modularity. We further investigated the entirety of all plasmids of each isolate (plasmidome) using a combination of short-read sequencing and machine-learning classifiers. Clustering of the plasmid sequences unraveled different E. faecium populations with a clear association with hospitalized patient isolates, suggesting different optimal configurations of plasmids in the hospital environment. The characterization of these populations allowed us to identify common mechanisms of plasmid stabilization such as toxin-antitoxin systems and genes exclusively present in particular plasmidome populations exemplified by copper resistance, phosphotransferase systems, or bacteriocin genes potentially involved in niche adaptation. Based on the distribution of k-mer distances between isolates, we concluded that plasmidomes rather than chromosomes are most informative for source specificity of E. faeciumIMPORTANCEEnterococcus faecium is one of the most frequent nosocomial pathogens of hospital-acquired infections. E. faecium has gained resistance against most commonly available antibiotics, most notably, against ampicillin, gentamicin, and vancomycin, which renders infections difficult to treat. Many antibiotic resistance traits, in particular, vancomycin resistance, can be encoded in autonomous and extrachromosomal elements called plasmids. These sequences can be disseminated to other isolates by horizontal gene transfer and confer novel mechanisms to source specificity. In our study, we elucidated the total plasmid content, referred to as the plasmidome, of 1,644 E. faecium isolates by using short- and long-read whole-genome technologies with the combination of a machine-learning classifier. This was fundamental to investigate the full collection of plasmid sequences present in our collection (pan-plasmidome) and to observe the potential transfer of plasmid sequences between E. faecium hosts. We observed that E. faecium isolates from hospitalized patients carried a larger number of plasmid sequences compared to that from other sources, and they elucidated different configurations of plasmidome populations in the hospital environment. We assessed the contribution of different genomic components and observed that plasmid sequences have the highest contribution to source specificity. Our study suggests that E. faecium plasmids are regulated by complex ecological constraints rather than physical interaction between hosts.

Serum and CSF soluble CD26 and CD30 concentrations in healthy pediatric surgical outpatients.

Activated T-helper type 1 (Th1) lymphocytes induce a cellular type immune response, and Th2 lymphocytes, a humoral or antibody-mediated type immune response. Soluble CD26 (sCD26) and soluble CD30 (sCD30) are regarded as markers of Th1 and Th2 lymphocyte activation, respectively. Serum from 112 generally healthy pediatric surgical patients and cerebrospinal fluid (CSF) from 39, aged 1-17 years were measured for sCD26 and sCD30 using an enzyme-linked immunosorbent assay method. The detection limit for sCD26 was 6.8 ng/ml and for sCD30, 1.9 IU/ml. For serum sCD26 and sCD30, 2.5% and 97.5% percentiles constituted the reference limits, and the 95% credible intervals for the percentiles were calculated using regression models with a Bayesian approach. A significant between-gender difference was observed (P = 0.015) in serum sCD26 concentration, of which the lower limits ranged between 273 and 716 ng/ml for girls and 235 and 797 ng/ml for boys. The upper limits ranged between 1456 and 1898 ng/ml for girls and between 1419 and 1981 ng/ml for boys. Moreover, the concentrations of sCD26 increased in infants and children up to 10 years in girls and 12 years in boys. After this however, the values decreased. The serum sCD30 concentration was highest among the youngest infants aged 1 year (80-193 IU/ml), after which a consistent age-related decrease was found. The lowest values were found at the age of 17 years (10-89 IU/ml). A significant between-gender difference in sCD30 concentration was observed (P = 0.019). sCD26 and sCD30 concentrations were low in the CSF samples analyzed: 13.3 ng/ml (median); range 8.3-51.5 ng/ml and 7.6 IU/ml; 2.1-18.5 IU/ml, respectively. Reference limits for serum sCD26 in children aged 1-17 years were established as being 235-1800 ng/ml in toddlers and 400-1800 ng/ml in female adolescents and 700-2000 ng/ml in male adolescents. For sCD30; reference limits of 80-190 IU/ml were established in the youngest age group and 10-90 IU/ml in adolescents.

Changes in MMP-2 and -9 activity and MMP-8 reactivity after amphotericin B induced synovitis and treatment with bufexamac.

The objective here was to evaluate the acute effects of induced arthritis on synovial fluid (SF) levels of matrix metalloproteinases (MMP) -2, -8 and -9 in horses. To evaluate MMP-2 and -9 activities and the effect of non-steroidal anti-inflammatory drug (NSAID) bufexamac during remission from acute arthritis. Aseptic arthritis was induced in 24 Standardbred horses using 20 mg of amphotericin B as a single intra-articular (IA) injection in the right intercarpal joint. After 1 week and 2 weeks, horses were treated intra-articularly with 10, 20, or 40 mg of bufexamac suspension or with sterile saline solution as control. SF was sampled prior to induction and at weekly intervals for 5 weeks. Fluids were evaluated for MMP-2 and MMP-9 activity by gelatin zymography or for MMP-8 immunoreactivity by Western Blotting. IA injection of amphotericin B consistently resulted in significant increase in the immunoreactivity of MMP-8 and activity of both the latent and the active forms of MMP-2 and -9, among which the active form of MMP-2 increased the most. MMP-9 levels declined to pre-induction levels within 2 weeks, whereas levels of MMP-2 remained still high after 5 weeks. Treatment with bufexamac did not significantly affect levels of gelatinolytic MMP. Results suggest that after acute arthritis of horses, elevated MMP activity is present in the joint, for several weeks, to a degree that could promote cartilage degradation, and treatment with the NSAID bufexamac is not likely to affect that. Furthermore, analysing levels of MMP-9 activity and especially levels of active forms of MMP-2 activity may be valuable to predict the time of occurrence of arthritis in horses.