Lactobacilli and Klebsiella: Two Opposites in the Fight for Human Health.

The problem of antibiotic resistance is currently very acute. Numerous research and development of new antibacterial drugs are being carried out that could help cope with various infectious agents. One of the promising directions for the search for new antibacterial drugs is the search among the probiotic strains present in the human gastrointestinal tract. This review is devoted to characteristics of one of these probiotic strains that have been studied to date: Limosilactobacillus reuteri. The review discusses its properties, synthesis of various compounds, as well as role of this strain in modulating various systems of the human body. The review also examines key characteristics of one of the most harmful among the currently known pathogenic organisms, Klebsiella, which is significantly resistant to antibiotics existing in medical practice, and also poses a great threat of nosocomial infections. Discussion of characteristics of the two strains, which have opposite effects on human health, may help in creation of new effective antibacterial drugs without significant side effects.

Study of the structure-function relationship of formate dehydrogenase- an important enzyme for Staphylococcus aureus biofilms by rational design.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Staphylococcus aureus (SauFDH) plays an important role in the vital activity of this bacterium, especially in the form of biofilms. Understanding its mechanism and structure-function relationship can help to find special inhibitors of this enzyme, which can be used as medicines against staphylococci. The gene encoding SauFDH was successfully cloned and expressed in our laboratory. This enzyme has the highest kcat value among the described FDHs and also has a high temperature stability compared to other enzymes of this group. That is why it can also be considered as a promising catalyst for NAD(P)H regeneration in the processes of chiral synthesis with oxidoreductases. In this work, the principle of rational design was used to improve SauFDH catalytic efficiency. After bioinformatics analysis of the amino acid sequence in combination with visualization of the enzyme structure (PDB 6TTB), 9 probable catalytically significant positions 119, 194, 196, 217-219, 246, 303 and 323 were identified, and 16 new mutant forms of SauFDH were obtained and characterized by kinetic experiments. The introduction of the mentioned substitutions in most cases leads to a decrease in stability at high temperatures and an increase at low temperatures. Substitutions in positions 119 and 194 lead to a decreasing of KMNAD+. A consistent decrease in the Michaelis constant in the Ile-Val-Ala-Gly series at position 119 of SauFDH is shown. KMNAD+ of mutant SauFDH V119G decreased by 27 times compared to the wild-type enzyme. After substitution Phe194Val KMNAD + decreased by 3.5 times. The catalytic constant for this mutant form practically did not change. For this mutant form, an increase in catalytic efficiency was demonstrated through the use of a multicomponent buffer system.

Ribonucleoside Hydrolases-Structure, Functions, Physiological Role and Practical Uses.

Ribonucleoside hydrolases are enzymes that catalyze the cleavage of ribonucleosides to nitrogenous bases and ribose. These enzymes are found in many organisms: bacteria, archaea, protozoa, metazoans, yeasts, fungi and plants. Despite the simple reaction catalyzed by these enzymes, their physiological role in most organisms remains unclear. In this review, we compare the structure, kinetic parameters, physiological role, and potential applications of different types of ribonucleoside hydrolases discovered and isolated from different organisms.

NAD+-Dependent Formate Dehydrogenase from Themotolerant Yeast Ogataea parapolymorpha: Properties and Protein Engineering of the N-Terminal Sequence.

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that addition of an Ala residue to the N-terminus reduces four-fold the rate constant of thermal inactivation compared with the addition of a Gly residue. Addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to acceleration of purification, practically does not affect kinetic parameters, but somewhat reduces thermal stability, which, however, can be restored to the level of OpaFDH_AK stability by adding 0.5 M NaCl.

Chimeric versus isolated proteins: Biochemical characterization of the NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 fused with the Baeyer-Villiger monooxygenase from Thermobifida fusca.

The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).

Speeding up SDS-PAGE: Theory and experiment.

In order to accelerate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), here we propose an optimized version of the technique enabled by experimental tuning reinforced by theoretical description. In the resulting system, the gel buffer was diluted twofold and supplemented with glycine at a low concentration, whereas a higher voltage was applied. This approach reduced runtime from 90 to 18 min. It is important to emphasize that, despite the high voltage applied to the gel, the resolution of the bands did not decrease compared to the original Laemmli method. The proposed acceleration approach can be used in other variants of SDS-PAGE.

Structure-Functional Examination of Novel Ribonucleoside Hydrolase C (RihC) from Limosilactobacillus reuteri LR1.

Ribonucleoside hydrolase C (RihC, EC 3.2.2.1, 3.2.2.2, 3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases Rih and catalyzes the cleavage of ribonucleosides to nitrogenous bases and ribose. RihC is one of the enzymes that are synthesized by lactobacilli in response to the presence of Klebsiella. To characterize this protein from Limosilactobacillus reuteri LR1, we cloned and expressed it. The activity of the enzyme was studied towards a wide range of substrates, including ribonucleosides, deoxyribonucleosides as well as an arabinoside. It was shown that the enzyme is active only with ribonucleosides and arabinoside, with the best substrate being uridine. The thermal stability of this enzyme was studied, and its crystal structure was obtained, which demonstrated the tetrameric architecture of the enzyme and allowed to shed light on a correlation between its structure and enzymatic activity. Comprehensive comparisons of all known RihC structures, both existing crystal structures and computed model structures from various species, were made, allowing for the identification of structural motifs important for enzyme functioning.

Efficient Assay and Marker Significance of NAD+ in Human Blood.

Oxidized nicotinamide adenine dinucleotide (NAD+) is a biological molecule of systemic importance. Essential role of NAD+ in cellular metabolism relies on the substrate action in various redox reactions and cellular signaling. This work introduces an efficient enzymatic assay of NAD+ content in human blood using recombinant formate dehydrogenase (FDH, EC 1.2.1.2), and demonstrates its diagnostic potential, comparing NAD+ content in the whole blood of control subjects and patients with cardiac or neurological pathologies. In the control group (n = 22, 25-70 years old), our quantification of the blood concentration of NAD+ (18 µM, minimum 15, max 23) corresponds well to NAD+ quantifications reported in literature. In patients with demyelinating neurological diseases (n = 10, 18-55 years old), the NAD+ levels significantly (p < 0.0001) decrease (to 14 µM, min 13, max 16), compared to the control group. In cardiac patients with the heart failure of stage II and III according to the New York Heart Association (NYHA) functional classification (n = 24, 42-83 years old), the blood levels of NAD+ (13 µM, min 9, max 18) are lower than those in the control subjects (p < 0.0001) or neurological patients (p = 0.1). A better discrimination of the cardiac and neurological patients is achieved when the ratios of NAD+ to the blood creatinine levels, mean corpuscular volume or potassium ions are compared. The proposed NAD+ assay provides an easy and robust tool for clinical analyses of an important metabolic indicator in the human blood.

Structure-Activity Relationships and Transcriptomic Analysis of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors.

To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and "branched-tail" oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors' IC50 values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure-activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 µM concentrations matching the effect of 30 µM roxadustat and 500 µM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.

Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity.

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.

Advantages of formate dehydrogenase reaction for efficient NAD+ quantification in biological samples.

The medical significance of NAD+-dependent metabolic regulation acquires increasing attention, demanding rapid and clinically feasible quantification of NAD+ in complex biological samples. Here we describe the usage of formate dehydrogenase for a straightforward and highly specific fluorometric assay of NAD+ in tissue extracts, not requiring chromatographic separation of nucleotides. The assay employs the irreversible reaction of formate oxidation coupled to NAD+ reduction, catalyzed by the enzyme which has high affinity and specificity to NAD+, and is stable under a variety of conditions. The assay reliably quantifies NAD+ in the methanol extracts of the rat brain cortex and mitochondria.

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion.

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.

The bacteriolytic activity of native and covalently immobilized lysozyme against Gram-positive and Gram-negative bacteria is differentially affected by charged amino acids and glycine.

The emergence of new antibiotic-resistant bacterial strains means it is increasingly important to find alternatives to traditional antibiotics, such as bacteriolytic enzymes. The bacteriolytic enzyme lysozyme is widely used in medicine as an antimicrobial agent, and covalent immobilization of lysozyme can expand its range of possible applications. However, information on the effect of such immobilized preparations on whole bacterial cells is quite limited. Here, we demonstrate the differential effects of glycine and charged (basic and acidic) amino acids on the enzymatic lysis of Gram-positive and Gram-negative bacteria by soluble and immobilized lysozyme. Glycine and basic amino acids (histidine, lysine, and arginine) significantly increase the rate of lysis of Gram-negative Escherichia coli cells in the presence of soluble lysozyme, but they do not substantially affect the rate of enzymatic lysis of Gram-positive Micrococcus luteus. Glutamate and aspartate significantly enhance enzymatic lysis of both E. coli and M. luteus. When using immobilized lysozyme, the effects of amino acids on the rate of cell lysis are significantly reduced. For immobilized lysozyme, the presence of an external diffusion mode on cell lysis kinetics at bacterial concentrations below 4 × 108 colony-forming units·mL-1 was shown. The broadening of the pH optimum of lysozyme activity after immobilization has been demonstrated for both Gram-positive and Gram-negative bacteria. The Michaelis constant (Km) values of immobilized lysozyme were increased by 1.5-fold for E. coli cell lysis and 4.6-fold for M. luteus cell lysis compared to soluble enzyme. A greater understanding of the effect of amino acids on the activity of native and immobilized lysozyme is important for both the development of new materials for medical purposes and elucidating the interaction of lysozyme with bacterial cells. Of particular interest is our finding that lysozyme activity against Gram-negative bacteria is enhanced in the presence of glycine and charged amino acids over a wide range of concentrations.

"Branched Tail" Oxyquinoline Inhibitors of HIF Prolyl Hydroxylase: Early Evaluation of Toxicity and Metabolism Using Liver-on-a-chip.

BACKGROUND: "Branched tail" oxyquinolines, and adaptaquin in particular, are potent HIF prolyl hydroxylase inhibitors showing promising results in in vivo hemorrhagic stroke models. The further improvement of the potency resulted in identification of a number of adaptaquin analogs. Early evaluation of toxicity and metabolism is desired right at the step of lead selection. OBJECTIVE: The aim of the study is to characterize the toxicity and metabolism of adaptaquin and its new improved analogs. METHOD: Liver-on-a-chip technology with differentiated HepaRG cells followed by LC-MS detection of the studied compounds and metabolites of the P450 substrate-inhibitor panel for CYP2B6, CYP2C9, CYP2C19, and CYP3A4. RESULTS: The optimized adaptaquin analogs show no toxicity up to a 100-fold increased range over EC50. The drugs are metabolized by CYP3A4 and CYP2B6 as shown with the use of the cytochrome P450 substrate-inhibitor panel designed and optimized for preclinical evaluation of drugs' in vitro biotransformation on a 3D human histotypical cell model using "liver-on-a-chip" technology. Activation of CYP2B6 with the drugs tested has been observed. A scheme for adaptaquin oxidative conversion is proposed. CONCLUSION: The optimized adaptaquin analogs are suitable for further preclinical trials. Activation of CYP2B6 with adaptaquin and its variants points to a potential increase in Tylenol toxicity if administered together.

L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?

L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40-100 µM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.

Bioactive Flavonoids and Catechols as Hif1 and Nrf2 Protein Stabilizers - Implications for Parkinson's Disease.

Flavonoids are known to trigger the intrinsic genetic adaptive programs to hypoxic or oxidative stress via estrogen receptor engagement or upstream kinase activation. To reveal specific structural requirements for direct stabilization of the transcription factors responsible for triggering the antihypoxic and antioxidant programs, we studied flavones, isoflavones and catechols including dihydroxybenzoate, didox, levodopa, and nordihydroguaiaretic acid (NDGA), using novel luciferase-based reporters specific for the first step in HIF1 or Nrf2 protein stabilization. Distinct structural requirements for either transcription factor stabilization have been found: as expected, these requirements for activation of HIF ODD-luc reporter correlate with in silico binding to HIF prolyl hydroxylase. By contrast, stabilization of Nrf2 requires the presence of 3,4-dihydroxy- (catechol) groups. Thus, only some but not all flavonoids are direct activators of the hypoxic and antioxidant genetic programs. NDGA from the Creosote bush resembles the best flavonoids in their ability to directly stabilize HIF1 and Nrf2 and is superior with respect to LOX inhibition thus favoring this compound over others. Given much higher bioavailability and stability of NDGA than any flavonoid, NDGA has been tested in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-animal model of Parkinson's Disease and demonstrated neuroprotective effects.

Engineering the pH-optimum of activity of the GH12 family endoglucanase by site-directed mutagenesis.

Endo-1,4-ß-glucanase from Penicillium verruculosum (PvEGIII) belongs to family 12 of glycoside hydrolases (GH12). Analysis of the enzyme 3D model structure showed that the amino acid residue Asp98 may directly affect the pH-profile of enzyme activity since it is located at the distance of hydrogen bond formation from Glu203 that plays the role of a general acid in catalysis. The gene encoding the PvEGIII was cloned into Escherichia coli. After the deletion of two introns, a plasmid construction was obtained allowing the PvEGIII expression in E. coli. Using site-directed mutagenesis, the Asp98Asn mutant of the PvEGIII was obtained. Both the wild type and mutant PvEGIIIs were expressed in E. coli with a yield of up to 1 g/L and then isolated in a highly purified form. The enzyme specific activity against soluble carboxymethylcellulose was not changed after a single amino acid substitution. However, the pH-optimum of activity of the mutant PvEGIII was shifted from pH 4.0 to 5.1, compared to the wild type enzyme. The shift in the enzyme pH-optimum to more neutral pH was also observed on insoluble cellulose, in the process of enzymatic depigmentation of denim fabric. Similar situation featuring the effect of the Asp/Asn residue, located near the Glu catalytic residue, on the enzyme activity pH-profile has previously been described for xylanases of the GH11 family. Thus, the glycoside hydrolases belonging to the GH11 and GH12 families function by a rather similar mechanism of catalysis.

Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutations.

The analysis of the 3D model structure of the ternary complex of recombinant formate dehydrogenase from soya Glycine max (EC 1.2.1.2., SoyFDH) with bound NAD+ and an inhibitor azide ion revealed the presence of hydrophobic Phe290 in the coenzyme-binding domain. This residue should shield the enzyme active site from solvent. On the basis of the alignment of plant FDHs sequences, Asp, Asn and Ser were selected as candidates to substitute Phe290. Computer modeling indicated the formation of two (Ser and Asn) or three (Asp) new hydrogen bonds in such mutants. The mutant SoyFDHs were expressed in Escherichia coli, purified and characterized. All amino acid substitutions increased K(м)(HCOO-) from 1.5 to 4.1-5.0 mM, whereas the K(м)(NAD+) values remained almost unchanged in the range from 9.1 to 14.0 µM, which is close to wt-SoyFDH (13.3 µM). The catalytic constants for F290N, F290D and F290S mutants of SoyFDH equaled 2.8, 5.1 and 4.1 s⁻¹, respectively; while that of the wild-type enzyme was 2.9 s⁻¹. The thermal stability of all mutant SoyFDHs was much higher compared with the wild-type enzyme. The differential scanning calorimetry data were in agreement with the results of thermal inactivation kinetics. The mutations F290S, F290N and F290D introduced into SoyFDH increased the T(m) values by 2.9°C, 4.3°C and 7.8°C, respectively. The best mutant F290D exhibited thermal stability similar to that of FDH from the plant Arabidopsis thaliana and exceeded that of the enzymes from the yeast Candida boidinii and the bacterium Moraxella sp. C1.

Isolation and preliminary characterization of new cytochrome c from autotrophic haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens.

New small cytochrome c (TniCYT) was purified from haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens. The protein was analyzed by mass spectrometry as well as using visible, CD and EPR spectroscopy. It was found that TniCYT is a monomer with a molecular mass of 9461 Da which contains two hemes per molecule. The data of CD and EPR spectroscopy showed that two hemes possess different optical activity and are in distinct, high and low spin states. TniCYT was also demonstrated to have unusual characteristics in the visible spectrum, namely, the splitting of characteristic peaks was observed in α- and ß-bands of the heme spectrum when the reduced form of cytochrome was analyzed. The α-band has two peaks with maximum at 548 and 556 nm whereas the ß-band showes ones at 520 and 528 nm. According to the MALDI finger-print analysis, the new cytochrome has a unique amino acid sequence.

Spread of vancomycin-resistant Enterococcus faecium in two haematological centres in Russia.

This paper describes the clonal diversity of vancomycin-resistant Enterococcus faecium isolated from patients with haematological malignancies in Russia. Pulsed-field gel electrophoresis (PFGE) typing of 129 vanA-positive E. faecium strains revealed 23 independent restriction profiles with two predominant clonal types. Multilocus sequence typing (MLST) of 16 strains selected from two predominant PFGE types showed that they belong to the epidemic clonal complex (CC) 17. Tn1546-like elements of isolates were compared with the prototype element from E. faecium BM4147 by polymerase chain reaction (PCR). Four different Tn1546 types were distinguished according to structural alternations. Polymorphism in the orf1 and vanSH genes was detected. However, a significant prevalence of the prototype Tn1546 was revealed. Tn1546-like elements with the same structures were observed in strains of different PFGE types. The virulence genes esp, gelE and hyl were detected by PCR in 118 isolates (91%), 87 isolates (67%) and 35 isolates (27%), respectively. In contrast, agg and cylA genes were not found. The detection frequency of esp was higher in epidemic strains than in sporadic ones (100% vs. 56%; P<0.05). This study describes a genetically variable population of vancomycin-resistant E. faecium in two Russian haematological centres. The spread of vancomycin resistance was mostly due to the distribution of the two subclones of E. faecium CC17, enriched with the virulence marker esp. At the same time, dissemination of an altered Tn1546 also occurred.