The rapid detection of procalcitonin in septic serum using immunoaffinity MALDI chips.

BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

Gut microbiome diversity of porcine peritonitis model of sepsis.

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.

Genomic characterisation of three GES-producing Enterobacterales isolated in the Czech Republic.

OBJECTIVES: The aim of the study is to characterise the genomic features of three GES-producing Enterobacterales isolates from Czech hospitals. METHODS: In 2020, during a routine screening of the hospital's surfaces in Prague General Hospital, two strains (CZ862 and CZ863) that belonged to the Enterobacter cloacae complex were found to be blaGES positive. Another blaGES positive strain identified as Klebsiella oxytoca was recovered from a patient hospitalised in Pilsen. Antibiotic susceptibility profiling was done with broth microdilution assay. Conjugation/transformation experiments were performed on all three strains. Genomic DNA of the three isolates was subjected to whole genome sequencing using PacBio platform. RESULTS: Multilocus sequence types typing of CZ862 and CZ863 identified the strains as ST837 and a novel ST (ST1622). Both blaGES harbouring plasmids showed high sequence similarity and complete query coverage (100% and 99.98%) with pEcl-35771cz. Both plasmids had two copies of blaGES instead of one copy as found in pEcl-35771cz. The clinical isolate CZ598 belonged to ST180. The plasmid harboured blaGES-7 gene, cat and aac(6')-lb and the novel variant blaOXA-1011. No similar sequences were observed, suggesting a novel plasmid. CONCLUSION: The detection of the two blaGES-positive plasmids in the same hospital environment, the first report after 3 years, suggests a hidden source. This highlights the importance of the hidden sources and evolution of such plasmids on the route of spreading into clinical settings. Also, the detection of the new blaOXA-1011, which is thought in this case to be associated with carbapenem resistance, imposes a health risk if disseminated, limiting therapeutic options.

Implication of different replicons in the spread of the VIM-1-encoding integron, In110, in Enterobacterales from Czech hospitals.

Background: VIM metallo-ß-lactamases are enzymes characterized by the ability to hydrolyze all ß-lactams. Usually, bla VIM-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 bla VIM genes. Materials and methods: 32 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. Results: The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the bla VIM-1 allele while six isolates carried the bla VIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The bla VIM-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 bla VIM 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 bla VIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. Conclusion: We 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.

Evidence of an epidemic spread of KPC-producing Enterobacterales in Czech hospitals.

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.

Genetic Plurality of OXA/NDM-Encoding Features Characterized From Enterobacterales Recovered From Czech Hospitals.

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 ß-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: bla NDM- 5 + bla OXA- 181; Kpn50595: bla NDM- 1 + bla OXA- 181; Kpn51015: bla NDM- 1 + bla OXA- 244; Eco52418: bla NDM- 5 + bla OXA- 244). In Kpn51015, the bla OXA- 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, bla OXA- 181 was identified on a ColKP3 plasmid in isolate Kpn47733, while a bla OXA- 181-carrying plasmid being an IncX3-ColKP3 fusion was identified in Kpn50595. The bla NDM- 1 gene was found on two different plasmids, p51015_NDM-1 belonging to a novel IncH plasmid group and p51015_NDM-1 being an IncF K 1-FIB replicon. Furthermore, the bla NDM- 5 was found in two IncFII plasmids exhibiting limited nucleotide similarity to each other. In both plasmids, the genetic environment of bla NDM- 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 bla CTX-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.

Detection of Five mcr-9-Carrying Enterobacterales Isolates in Four Czech Hospitals.

The aim of this study was to report the characterization of the first mcr-positive Enterobacterales isolated from Czech hospitals. In 2019, one Citrobacter freundii and four Enterobacter isolates were recovered from Czech hospitals. The production of carbapenemases was examined by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay. Additionally, bacteria were screened for the presence of carbapenemase-encoding genes and plasmid-mediated colistin resistance genes by PCR. To define the genetic units carrying mcr genes, the genomic DNAs of mcr-carrying clinical isolates were sequenced on the PacBio Sequel I platform. Results showed that all isolates carried blaVIM- and mcr-like genes. Analysis of whole-genome sequencing (WGS) data revealed that all isolates carried mcr-9-like alleles. Furthermore, the three sequence type 106 (ST106) Enterobacter hormaechei isolates harbored the blaVIM-1 gene, while the ST764 E. hormaechei and ST95 C. freundii included blaVIM-4 Analysis of plasmid sequences showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. Additionally, at least one multidrug resistance (MDR) region was identified in each mcr-9-carrying IncHI2 plasmid. The blaVIM-4 gene was found in the MDR regions of p48880_MCR_VIM and p51929_MCR_VIM. In the three remaining isolates, blaVIM-1 was localized on plasmids (∼55 kb) exhibiting repA-like sequences 99% identical to the respective gene of pKPC-CAV1193. In conclusion, to the best of our knowledge, these 5 isolates were the first mcr-9-positive bacteria of clinical origin identified in the Czech Republic. Additionally, the carriage of the blaVIM-1 on pKPC-CAV1193-like plasmids is described for the first time. Thus, our findings underline the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants.IMPORTANCE 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. Here, we report the first detection of Enterobacterales carrying mcr-9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 Enterobacter hormaechei isolates harbored blaVIM-1, while the ST764 E. hormaechei and ST95 Citrobacter freundii isolates included blaVIM-4 Analysis of WGS data showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. blaVIM-4 was found in the MDR regions of IncHI2 plasmids, while blaVIM-1 was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms.

Insufficient repeatability and reproducibility of MALDI-TOF MS-based identification of MRSA.

Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is essential for proper initial antibiotic therapy and timely set up of hygienic measures. Recently, detection of MRSA using MALDI-TOF mass spectrometer mediated by the peptide-phenol-soluble modulin (PSM-mec)-linked to the class A mec gene complex present in SCCmec cassettes types II, III, and VIII of MRSA strains, has been commercially available. We present here a multicentre study on MALDI-TOF MS detection of MRSA evincing a poor repeatability and reproducibility of the assay. The sensitivity of the assay varies between 50 and 90% in strains carrying psmMEC and psmδ genes encoding for PSM-mec and δ-toxin (a member of the PSM peptide family), respectively. No false positive results were found. The very major error calculation was 30% and the major error achieved 0%. Interlaboratory repeatability varies between 0 and 100%. No significant difference was observed with the use of different cultivation media. Our data showed a poor sensitivity of the method excluding it from the use in routine laboratory testing.