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Objective. To study virulence genes in E. faecium and E. faecalis isolated from the blood cultures of patients with hematological diseases. Materials and Methods. Virulence genes were studied in E. faecium and E. faecalis strains isolated from blood culture from hematological patients in four Russian hospitals (2002–2020). Susceptibility to vancomycin was determined by broth microdilution method (CLSI, 2022). Virulence genes (esp, hyl, asa1, cylA and gelE) in E. faecium and E. faecalis as well as vancomycin resistance genes (vanA, vanB and vanD) in Enterococcus spp. were detected by multiplex PCR. Results. A total of 551 Enterococcus spp. strains were studied, of them 440 (79.9%) were E. faecium and 111 (20.1%) E. faecalis. Resistance to vancomycin was detected in 86 (19.5%) E. faecium, of them 62 (72.1%) carried vanA and 24 (27.9%) vanB genes. One (1.1%) of 111 E. faecalis was vancomycinintermediate (MIC 16 μg/ml) with vanD gene. Virulence genes were detected in 86.2% of Enterococcus spp., significantly more often among E. faecalis (95.5%) compared to E. faecium (83.9%, p = 0.003). The predominant genes in E. faecium were esp (70.2%) and hyl (52.1%), the detection of the asa1, cylA and gelE genes was minimal. Other genes dominated in E. faecalis: gelE (66.7%), asa1 (65.8%), cylA (36.9%). Statistically significant differences between E. faecium and E. faecalis were determined for all studied virulence genes (p < 0.0001). A combination of three or more virulence genes was detected significantly more often among E. faecalis in comparison with E. faecium (45% vs. 2.5%, p < 0.0001), whereas the presence of one gene or their absence prevailed in E. faecium (40.2% vs. 17.1%, p < 0.0001; 16.1% vs. 4.5%, p = 0.003, respectively). When comparing the two study periods (2002–2010 and 2011–2020), E. faecalis showed a significant increase in the proportion of strains with a combination of three or more virulence genes from 33.3% to 55% (p = 0.03), in E. faecium – with one virulence gene (from 31.7% to 46.3%, p = 0.002) and no genes (from 6.6% to 23%, p < 0.0001). Conclusions. Differences in the frequency of detection of virulence genes and their spectrum have been identified between E. faecium and E. faecalis strains. Virulence genes are significantly more often detected in E. faecalis with a predominance of combinations of three or more genes.

Objective. To present the results of using in-house method for rapid identification of fungi from funguspositive bottles with routine conventional culture-based identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in patients with bloodstream infection. Materials and Methods. Prospective study was performed from 2016 to 2019 at the National Research Center for Hematology, Moscow. During the study period, all blood cultures (BC) bottles obtained from hematological patients were incubated in the BACTEC FX system (Becton Dickinson, USA). Positive BC bottles were examined by Gram stain. In house method was used after Gram stain was positive for yeast cells or hyphae. For that, BC media was transfer from fungus-positive bottles into tube. In house method included series section steps consisted from centrifugation and extraction of fungal proteins by adding of sodium dodecyl sulfate. Routine conventional culture-based identification on Sabouraud with chloramphenicol agar (bioMerieux, France) for yeasts and on Sabouraud dextrose agar (Oxoid, UK) for molds was used simultaneously with the in house method. Results. During the study period, 16 fungus-positive bottles were obtained from which were isolated in monoculture 14 (87.5%) Candida spp.: C. parapsilosis (n = 5), C. tropicalis (n = 4), C. albicans (n = 3), C. krusei (n = 1), C. guilliermondii (n = 1), one (6.3%) Rhodotorula mucilaginosa and one (6.3%) Fusarium dimerum. The in house method resulted in 75% (12/16) and 68.8% (11/16) identification rate at the genus and species level of fungi, respectively. The identification of fungi to species level was confirmed by conventional culture-based method in all cases. The median time from the start of vial incubation in BACTEC FX system to identification of fungi by in house method was less than conventional culture-based identification: 36 hrs 20 min vs 55 hrs 31 min (p = 0.028). Conclusions. A high rate of correct direct species identification and significant reduction in time to verification of fungi from fungus-positive bottles by in house method were obtained. The proposed in house method should be recommended for use in real microbiology practice to reduce the time for submitting results of identification to clinical units.

Objective. To study the genetic diversity of vancomycin-resistant Enterococcus faecium (VR-E. faecium) isolated from the blood culture in patients with hematological malignancies by multilocus sequence typing (MLST). Materials and Methods. VR-E. faecium isolated from the blood culture in hematological patients in 6 hospitals of 4 Russian cities (2003–2019) were evaluated. Susceptibility to vancomycin was tested by the broth microdilution method (CLSI, 2018). Vancomycin-resistance genes (vanA, vanB) were identified by polymerase chain reaction. Genotyping of VR-E. faecium was performed by MLST. Results. A total of 83 VR-E. faecium were examined. The vanA genes were detected in 71.1% (n = 59) VR-E. faecium, vanB genes – in 28.9% (n = 24). A total of 22 sequence types (STs) belonging to epidemic clonal complex CC17 were detected. The dominant sequence types were ST17 (19.3%), ST78 (18.1%), ST80 (16.9%), and comprised 54.3% VR-E. faecium. Other sequence types included 1 to 4 strains. VR-E. faecium carrying vanA, in comparison with VR-E. faecium vanB, significantly more often belonged to ST78 (23.7% vs. 4.2%, p = 0.0559, respectively) and ST80 (23.7% versus 0%, p = 0.0079, respectively) and less frequently to ST17 (6,8% versus 50%, р < 0.0001). Circulation of 9 STs including «high-risk» clones ST17 and ST78 was detected during two study periods (2003–2011 and 2012–2019). Conclusions. This study showed a genetic diversity of VR-E. faecium that was represented by 22 STs. All VR-E. faecium belonged to epidemic clonal complex CC17 and comprised «high-risk» clones ST17, ST78 and less common STs.

Objective. To evaluate the prevalence of acquired carbapenemase genes among A. baumannii isolates collected from blood culture in patients with hematological malignancies. Materials and Methods. This prospective multicenter study included A. baumannii isolated from blood culture in hematological patients in 7 Russian hospitals (2003–2015). Minimum inhibitory concentrations (MICs) of meropenem and imipenem were determined by broth microdilution method (CLSI, 2017). All A. baumannii isolates were tested by multiplex real-time PCR for presence of acquired carbapenemase genes (blaOXA-23-like, blaOXA-24/40-like, blaOXA-58-like, blaNDM-like, blaVIM-like and blaIMP-like). Results. A total of 74 A. baumannii isolates were studied, of them 55 (74.3%) were non-susceptible to meropenem and/or imipenem. Genes of acquired OXA-carbapenemases were detected in 70.9% (39/55) of carbapenem non-susceptible isolates. Metallo-beta-lactamase genes (blaNDM-like, blaVIM-like and blaIMP-like) were not detected. The most prevalent carbapenemase genes were blaOXA-24/40-like (51.3%), followed by blaOXA-23-like (38.5%) and blaOXA-58-like (10.3%). The MIC50/90 values of meropenem and imipenem were higher in carbapenem non-susceptible A. baumannii harboring carbapenemase genes compared to isolates without acquired carbapenemase genes. A. baumannii isolates carrying blaOXA-24/40-like demonstrated the highest MIC50/90 values of carbapenems. Conclusions. The majority of carbapenem non-susceptible A. baumannii isolates (70.9%) carried acquired carbapenemase genes, of which blaOXA-24/40-like were predominant. The highest MIC50/90 values of carbapenems were detected in A. baumannii harboring blaOXA-24/40-like.

Objective. The objective of the study was to evaluate the prevalance of genes blaTEM, blaSHV and blaCTX-M among extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales isolates collected from blood culture in patients with hematological malignancies. Materials and methods. The prospective multicenter study included Enterobacterales isolates collected from blood culture in hematological patients from 10 Russian hospitals (2003-2015). The isolates were screened for ESBLs by the phenotypic methods (CLSI 2017). Genes encoding TEM, SHV and CTX-M were detected using polymerase chain reaction (PCR) by specific primers. Results. A total of 499 ESBL-producing Enterobacterales were evaluated. Among them Klebsiella pneumoniae (43.9%) and Escherichia coli (43.3%) predominated. The tested genes were present in combination (79%) and alone (19.6%). Genes of blaCTX-M were most common (82.6%), followed by blaTEM (73.7%) and blaSHV (53.7%). The genes (blaTEM, blaSHV and blaCTX-M) were not detected in 1.4% (n=7) ESBLproducing Enterobacterales. Genes blaCTX-M were in 84.9% K. pneumoniae, 89.8% E. coli and 42.6% Enterobacter cloacae. The blaCTX-M genes belonged to the subtypes blaCTX-M-1 (88.8%), blaCTX-M-9 (14.8%) and blaCTX-M-2 (0.2%). Two different subtypes of blaCTX-M were in 4.1% Enterobacterales isolates. Conclusions. Among the ESBL-producing Enterobacterales isolates blaCTX-M genes were most common (82.6%), the majority of them belonged to the blaCTX-M-1 subtypes (88.8%). Most of Enterobacterales isolates (79%) harbored several types of beta-lactamases genes and 4.1% isolates carried two subtypes of blaCTX-M.

Incidence, severity, and risk factors for hemorrhagic cystitis (HC) were assessed in 267 patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). HC was diagnosed in 14.6% (39 patients) within 1-139 days after allo-HSCT (median duration – 39 days). Chemotherapy-related HC was diagnosed in 4 patients only. The majority (19/35) of patients developed late HC of viral aetiology. Median time from a day of HC diagnosis to clinical symptoms resolution was 25 days (range: 6 to 133 days). Using a multivariate analysis, allo-HSCT from mismatched unrelated/haploidentical donor was found to be a risk factor of HC (р=0.01). The analysis also showed that 82.1% of patients with HC received cyclophosphamide as a part of conditioning regimen or +3/+4 days after allo-HSCT.

Objective. To evaluate the biofilm formation by isolates of Candida spp. obtained from blood culture in patients with or without hematological malignancies. Materials and Methods. Biofilm formation by Candida spp. was determined by the method of Pierce C. and Tumbarello M. Isolates with optical density (OD) >0.1 were considered as biofilm-forming. Results. A total of 109 isolates of Candida spp. (C. albicans [n=22], C. parapsilosis [n=22], C. tropicalis [n=22], C. krusei [n=21], C. glabrata [n=22]) obtained from patients with or without hematological malignancies (55 and 54 isolates, respectively) were studied. Biofilm formation was determined in 54% of Candida spp. isolates (n=59). Biofilm production was observed more often in non-albicans species compared to C. albicans (60% versus 32%, p=0.02). The incidence of biofilm formation was significantly higher in isolates of C. tropicalis (82%) and C. krusei (81%) compared to isolates of C. parapsilosis (50%), C. albicans (32%), and C. glabrata (27%) (p

Objective. To evaluate antimicrobial susceptibility of Enterococcus spp. isolated from blood culture in patients with hematological malignancies. Materials and Methods. Antimicrobial susceptibility of 427 Enterococcus spp. collected from 10 hospitals in 8 cities of Russia in 2002-2016 as part of the multicenter study was tested by the broth microdilution method [CLSI 2015]. Results. Among bloodstream pathogens there was a prevalence of E. faecium (78.2%), followed by E. faecalis (19.7%) and other Enterococcus spp. (2.1%). A total of 50 (15%) vancomycin-resistant E. faecium (Vancomycin-resistant Enterococcus – VRE) was detected, of them 33 (66%) were harboring vanA gene, 17 (34%) – vanB gene. In 2012 one linezolid resistant E. faecium (MIC = 16 µg/mL) was detected. Linezolid-resistant E. faecium contained the G2576T 23S rRNA mutation. All VRE faecium including the one linezolid-resistant isolate were susceptible to daptomycin. All E. faecium were susceptible to tigecycline, 78.7% – to chloramphenicol, 74.9% – to tetracycline. Proportion of E. faecium with high level resistance to gentamicin was 85%, to streptomycin – 60%, to both aminoglycosides – 45%. All E. faecalis were susceptible to linezolid, teicoplanin and tigecycline; 97.6% – to ampicillin. One isolate of E. faecalis (1.2%) with intermediate susceptibility to vancomycin (MIC = 16 µg/mL) harboring vanD gene and one isolate of E. gallinarum resistant to vancomycin, carrying vanC1 and vanB genes were detected. Conclusions. Isolates of E. faecalis had more favorable profile of antimicrobial susceptibility comparing to E. faecium. A total of 15% E. faecium were vancomycin resistant and one of them had resistance to linezolid. In this study one E. faecalis and one E. gallinarum isolates were non-susceptible to vancomycin.

Objective. To detect vancomycin-resistant enterococci (VRE) using chromogenic selective medium CHROMagar™VRE (CHROMagar, France). Materials and Methods. In the first part of the study, a total of 39 vancomycin-resistant and 20 vancomycinsusceptible Enterococcus spp. isolated from blood culture with known susceptibility profiles were incubated on the CHROMagar™VRE (CHROMagar, France) and examined after 24 h and 48 h of incubation. In the second part of the study, a total of 110 rectal swabs were taken from patients with hematological malignancies and incubated on the CHROMagar™VRE. The vancomycin susceptibility of isolates grown on the selective medium was further evaluated by the broth microdilution method (CLSI, 2017). Glycopeptide resistance genes were detected by PCR. Results. Using the CHROMagar™VRE, a total of 36 (92.3%) vancomycin-resistant isolates were detected after 24 h and additional two isolates – after 48 h of incubation. The sensitivity of the selective medium for detection of VRE obtained from blood culture was 92% and 97% after 24 h and 48 h of incubation, respectively. All 20 vancomycin-susceptible enterococci did not grow on the CHROMagar™VRE (specificity – 100%). Of 110 rectal swabs, 35 (31.8%) samples were positive for Enterococcus spp. on the CHROMagar™VRE (33 – E. faecium и 2 – E. faecalis). Resistance to vancomycin was detected in 32/33 (97%) E. faecium isolates, of them 28 and 4 strains were isolated after 24 h and 48 h of incubation; all VRE strains carried vanA gene. The proportion of false positive isolates was 3.4% after 24 h of incubation and 8.6% after 48 h of incubation on the CHROMagar™VRE medium for screening of VRE from rectal swabs. Conclusions. The chromogenic selective media CHROMagar™VRE has a high sensitivity and specificity for the detection of VRE and can be used for screening in laboratory practice.

Objective. The objective of the study was to evaluate the prevalence of Clostridium difficile-associated diarrhea (CDAD) among hospitalized patients with antibiotic-associated diarrhea (AAD) in general and by specific types of medical care and hospital units. Materials and Methods. A prospective, cross-sectional, non-interventional, multicenter study. The main inclusion criteria were: patient age of 18 years and over, hospital stay of at least 48 hours, current antibiotic therapy or antibiotic therapy within the previous 30 days, loose stools (Bristol stool types 5-7 and stool frequency ≥3 within ≤24 consecutive hours or exceeding normal for the patient), and signed informed consent form. The stool sample was transported to the local (study site) microbiology laboratory for detection of glutamate dehydrogenase (GDH) and toxins A/B using enzyme immunoassay (EIA) stool test. Results. From April 2016 through April 2017, a total of 1,245 patients from 12 large hospitals were enrolled into the study. Data on 81 patients were excluded from the analysis due to the different reasons. Data on 1,164 patients (45.2% males and 54.8% females) with the mean age of 54.9 years (range: 18 to 95 years) were analyzed. Length of hospitalization was 2 to 188 days (median of 8 days). The EIA stool test showed CDAD positive results in 21.7% (253/1,164) patients. The patients were from surgery units (546/1,164), internal medicine units (510/1,164) and intensive care units (108/1,164). The prevalence of CDAD among patients from surgery, internal medicine and intensive care units was 26.2%, 17.8% and 17.6%, respectively. Oncology, gastroenterology, septic surgery, oncohaematology and general profiles of hospital units accounted for more than 75% of all patients included; the prevalence of CDAD by those hospital units was 11.3%, 15.0%, 39.2%, 17.6%, and 27.2%, respectively. The proportion of GDH-positive and toxin A/B-negative patients by the rapid stool test result was 16.8% (196/1,164). The prevalence of CDAD varied widely between the hospitals (from 0% to 44.3%). Conclusions. The prevalence of CDAD among hospitalized patients with AAD in this study was 21.7% (95% confidence interval: 14.8%, 28.7%). The percentage of CDAD varied widely between hospitals and by specific types of medical care and hospital units