Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the “last-resort” antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr, in different host species and geographic areas were published.

The aim of this study was to characterize four Enterobacterales co-producing NDM- and OXA-48-like carbapenemases from Czech patients with travel history or/and previous hospitalization abroad. Klebsiella pneumoniae isolates belonged to “high risk” clones ST147, ST11, and ST15, while the Escherichia coli isolate was assigned to ST167. All isolates expressed resistance against most β-lactams, including carbapenems, while retaining susceptibility to colistin. Furthermore, analysis of WGS data showed that all four isolates co-produced OXA-48- and NDM-type carbapenemases, in different combinations (Kpn47733: blaNDM–5 + blaOXA–181; Kpn50595: blaNDM–1 + blaOXA–181; Kpn51015: blaNDM–1 + blaOXA–244; Eco52418: blaNDM–5 + blaOXA–244). In Kpn51015, the blaOXA–244 was found on plasmid p51015_OXA-244, while the respective gene was localized in the chromosomal contig of E. coli Eco52418. On the other hand, blaOXA–181 was identified on a ColKP3 plasmid in isolate Kpn47733, while a blaOXA–181-carrying plasmid being an IncX3-ColKP3 fusion was identified in Kpn50595. The blaNDM–1 gene was found on two different plasmids, p51015_NDM-1 belonging to a novel IncH plasmid group and p51015_NDM-1 being an IncFK1-FIB replicon. Furthermore, the blaNDM–5 was found in two IncFII plasmids exhibiting limited nucleotide similarity to each other. In both plasmids, the genetic environment of blaNDM–5 was identical. Finally, in all four carbapenemase-producing isolates, a diverse number of additional replicons, some of these associated with important resistance determinants, like blaCTX–M–15, arr-2 and ermB, were identified. In conclusion, this study reports the first description of OXA-244-producing Enterobacterales isolated from Czech hospitals. Additionally, our findings indicated the genetic plurality involved in the acquisition and dissemination of determinants encoding OXA/NDM carbapenemases.

The aim of the present study is to describe the ongoing spread of the KPC-producing strains, which is evolving to an epidemic in Czech hospitals. During the period of 2018–2019, a total of 108 KPC-producing Enterobacterales were recovered from 20 hospitals. Analysis of long-read sequencing data revealed the presence of several types of blaKPC-carrying plasmids; 19 out of 25 blaKPC-carrying plasmids could be assigned to R (n = 12), N (n = 5), C (n = 1) and P6 (n = 1) incompatibility (Inc) groups. Five of the remaining blaKPC-carrying plasmids were multireplicon, while one plasmid couldn’t be typed. Additionally, phylogenetic analysis confirmed the spread of blaKPC-carrying plasmids among different clones of diverse Enterobacterales species. Our findings demonstrated that the increased prevalence of KPC-producing isolates was due to plasmids spreading among different species. In some districts, the local dissemination of IncR and IncN plasmids was observed. Additionally, the ongoing evolution of blaKPC-carrying plasmids, through genetic rearrangements, favours the preservation and further dissemination of these mobile genetic elements. Therefore, the situation should be monitored, and immediate infection control should be implemented in hospitals reporting KPC-producing strains.

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Antimicrobial resistance (AMR) currently represents a concern for human health, and the reintroduction of antibiotics such as fosfomycin into clinical practice can provide further option in treatment of multidrug-resistant (MDR) bacterial infections. However, there is a global increase of fosfomycin-resistant bacteria, reducing its effectiveness.

Advances in single cell analysis, especially cytometric approaches, have profoundly innovated immunological research. This has resulted in an expansion of high dimensional data, posing great challenges for comprehensive and unbiased analysis. Conventional manual analysis thus becomes untenable, while most computational methods lack flexibility and interoperability, hampering usability. Here, for the first time, we adapted Seurat, a single cell RNA sequencing (scRNA-seq) analysis package, for end-to-end flow cytometric data analysis. We showcased its robust analytical capacity by analyzing the adult blood and cord blood T cell profiles, which was validated by Spectre, another cytometric data analysis package, and manual analysis. Importantly, a unique CD8+CD45RA+CD27+CD161+T cell subset, was identified in cord blood and characterized using flow cytometry and scRNA-seq analysis from a published dataset. Collectively, Seurat possesses great potential for cytometric data analysis. It facilitates thorough interpretations of high dimensional data using a single pipeline, implementing data-driven investigation in clinical immunology.

Animal models are essential in understanding of the mechanisms of sepsis moreover the development and the assessment of emerging therapies. In clinically relevant porcine model, however, a significant variability in the host response has been observed among animals. Thus, there is a strong demand to better understand the potential sources of this heterogeneity. In this study, we compared faecal microbiome composition of 12 animals. Three samples were collected at different time points from each animal. Bacteriome was subjected to 16S rDNA profiling. A significant difference in bacterial composition was associated with the season (p < 0.001) but not with the sex of the pig (p = 0.28), the timing of sample collection (p = 0.59), or interactions thereof (all p > 0.3). The season batch explained 55% of the total variance in the bacteriome diversity. The season term was highly significant from the high-resolution level of the bacterial amplicon sequencing variants up to the level of phylum. The diversity of the microbiome composition could significantly influence experimental model of sepsis, and studies are warranted to demonstrate the effects of gut microbiome diversity on the host-response. If confirmed, control of the gut microbiome should become a standard part of the pre-clinical sepsis experiments.

BackgroundVIM metallo-β-lactamases are enzymes characterized by the ability to hydrolyze all β-lactams. Usually, blaVIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM-1 carbapenemase-encoding integron In110. However, during 2019–2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of blaVIM genes.Materials and methods32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform.ResultsThe 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the blaVIM-1 allele while six isolates carried the blaVIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The blaVIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the blaVIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two blaVIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome.ConclusionWe observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution.