Background: Fecal screening of patients for vancomycin-resistant enterococci (VRE) is recommended in an attempt to establish infection control measures. Culture-based methods for VRE detection are widely used, but particular attention should be paid to correct identification of growing isolates. This study is focused on species identification and antimicrobial susceptibility of vancomycin-resistant enterococci and other Gram-positive catalase-negative cocci, recovered from VRE screen cultures.Materials and methods: A total of 109 immunocompromised patients from University Multiprofile Hospital for Active Treatment "Dr G. Stranski", Pleven were screened for VRE. The determination of cocci was relied on cultural characteristics, manual and automated systems for identification as well as data on antimicrobial sensitivity.Results: A total of 57 Gram-positive catalase-negative cocci were isolated: 32 VRE, 11 vancomycin-susceptible enterococci and 14 enterococcus-like organisms. Colonization with VanA or VanC enterococci was detected in 29.35% of the patients, with a distinct prevalence of VanC (23.85%) over than VanA (5.50%). Six enterococci were confirmed as vanA genotype -5 E. faecium and 1 E. gallinarum. All E. faecium isolates expressed high-level resistance to vancomycin (MICs ≥256 µg/ml) and low-level resistance to teicoplanin (MICs: 4.0-6.0 µg/ml), whereas a single E. gallinarum isolate showed MICs ≥256 µg/ml for both glycopeptides. The isolated VanC enterococci (13 E. gallinarum and 13 E. casseliflavus) were susceptible to tested antibiotics and possessed low-level resistance to vancomycin (MICs: 4-12 µg/ml). Most of the recovered enterococcus-like organisms were identified as Leuconostoc spp.In conclusion, both species identification and antimicrobial susceptibility pattern have to be taken into account for distinguishing VRE and other Gram-positive catalase-negative cocci, growing in VRE screen cultures.

Background: The precise identification of yeasts and their antifungal sensitivity is a key factor in the choice of a suitable drug for treatment and prevention of fungal infections. Objectives: The aim of the present study was to determine in vitro susceptibility of clinical Candida albicans isolates to nine antifungal agents. Methods: A total of 61 C. albicans isolates were tested. Antifungal susceptibility was evaluated by determination of minimum inhibitory concentrations (MIC). Results: Fifty C. albicans isolates were susceptible to nine antifungal agents. The remaining 11 yeasts were resistant to one or more antifungals. All C. albicans were susceptible to amphotericin B with MICs 0.25 to 1 µg/mL and exhibited sensitivity to 5-fluorocytosine with MIC90 of 0.125 µg/mL. The MIC50 and MIC90 of fluconazole were 0.5 and 32 µg/mL, whereas the MIC50 and MIC90 of voriconazole were considerably lower-0.0078 and 2 µg/mL. The isolates showed susceptibility to echinocandins with MIC90 of micafungin, anidulafungin and caspofungin of 0.015, 0.031, and 0.125 µg/mL, respectively. Of particular interest was detection of seven C. albicans isolates, which expressed a high-level resistance to all azoles and one of them was also resistant to echinocandins. Conclusions: In conclusion, detection of resistance in C. albicans, which is a species typically susceptible to antifungals, is of great importance for clinical practice.

Background: Vancomycin-resistant enterococci (VRE) are recognized as nosocomial pathogens with increased importance in recent years. These bacteria are frequently isolated from patients admitted to intensive care units (ICUs). Enterococcal pathogenicity is enhanced by different antibiotic resistance and virulence determinants. Objectives: The present study aimed to assess the prevalence of genes encoding resistance to antibiotics and virulence factors in intestinal VRE isolates from ICU patients. Methods: In this study, 23 VREs were investigated. Minimum inhibitory concentrations (MICs) to nine antimicrobial agents were examined using E-test. Genes encoding vancomycin resistance (vanABCDMN), aminoglycoside-modifying enzymes (aac(6')-Ie-aph(2")-Ia, aph(2")-Ib, aph(2")-Ic, aph(2")-Id, aph(3')-IIIa, ant(3')-Ia, ant(4')-Ia, ant(6')-Ia), together with genes for various virulence factor (ace/acm, asa1, cylA, efaA, esp, gelE and hyl), were detected using multiplex PCR. Results: The species distribution of the tested VRE was as follows: Nine Enterococcus casseliflavus, seven E. gallinarum, and seven E. faecium. The vanA gene was found in all E. faecium, in six of which the classical VanA phenotype was observed. The vancomycin (vanC) phenotype was associated with the presence of vanC1 gene in E. gallinarum and the vanC2 gene in E. casseliflavus isolates. The aac(6')-Ie-aph(2")-Ia gene was encoding high-level gentamicin resistance (HLGR) in the studied VRE. All E. faecium were positive for acm and esp, while acm in combination with esp or hyl was detected in 2 vanC enterococci. Conclusions: According to the findings, there was a correlation between the phenotype and the genotype of glycopeptide resistance in the tested VRE. HLGR was more prevalent in E. faecium because of the presence of aac(6')-Ie-aph(2")-Ia. The higher prevalence of virulence determinants was confirmed in vanA isolates compared to the studied vanC-carrying enterococci.

Objective We conducted a study on Haemophilus influenzae isolates recovered from children with acute otitis media (AOM). We aimed to establish the distribution of noncapsulated (also known as nontypeable Haemophilus influenzae [NTHi]) and encapsulated H. influenzae in the study population, and the antimicrobial susceptibilities of the isolates. Methods We collected 113 nasopharyngeal swabs and 91 middle ear fluids/otorrhea specimens from patients up to 9 years of age with AOM. Of these, 26.1% (n = 53) were culture-positive for H. influenzae. Only one episode of AOM was included per patient. Conventional tests and rapid panel Neisseria/Haemophilus panel were used for the identification of the isolates. Detection of encapsulated and noncapsulated strains was done by polymerase chain reaction (PCR) for bexA gene. PCR-serotyping was performed for capsule types: “a” and “f.” Biotypes were assigned based on the indole, urease, and ornithine decarboxylase activity. Susceptibility testing was performed according to the criteria of European Committee on Antimicrobial Susceptibility Testing (EUCAST). Results Capsule determination showed that 96.2% of H. influenzae isolates responsible for “mild” and “severe” AOM cases in children were NTHi. Biotype I was predominantly associated with AOM isolates. Capsule types “a” and “c” were found in two isolates. Antibiotic resistance was found in 39.6% of the isolates. The highest resistance rate was for trimethoprim-sulfamethoxazole (37.7%). About 20.7% of isolates were ampicillin-resistant: 5.6% expressed a β-lactamase, and 15.1% had a β–lactamase-negative ampicillin-resistant phenotype. Conclusion The current prevalence rates of nonsusceptible H. influenzae to ampicillin appear to be low among AOM. NTHi is an emergent pathogen in AOM cases. Ongoing observations are needed about how NTHi colonizes, survives, and evolves into a leading causative agent of H. influenzae diseases.

Vancomycin-resistant enterococci (VRЕ) are recognized as important hospital pathogens which have become common in patients admitted to the intensive care units (ICUs). The purpose of this study was to evaluate the incidence of and the risk factors for colonization with VRE among ICU patients. A total of 91 patients who had duration of hospitalization more than 48 h and without infection caused by VRE or/and other microorganisms in the ICU at University Hospital, Pleven were screened for colonization with VRE. The following data were collected: demographic characteristics, clinical information and antimicrobials use. The statistical analysis was performed using SPSS version 27.0. Colonization with VRE was established in 22 patients and one was carrying two enterococcal species. A total of 23 VRE were isolated. The univariate analysis showed that the postoperative critical cares (p < 0.001), cardiovascular diseases (p = 0.009) and the presence of an endotracheal tube (p = 0.003) were risk factors for colonization with VRE. Also, the postoperative critical cares (p = 0.021) and cardiovascular diseases (p = 0.018) were confirmed as independent risk factor for VRE acquisition by multivariate analysis. The prevalence of VRE colonization among the ICU patients was relatively high (24.2%). Risk factors for acquisition of intestinal VRE were the postoperative cares, cardiovascular diseases and the presence of an endotracheal tube.

Background: Emerging non-albicans Candida (NAC) species are a major threat because of their intrinsic or acquired resistance to routinely applied antifungal agents. Objectives: The purpose of our study was to reveal in vitro activity of nine antifungal agents against NAC isolates. Methods: A total of 67 NAC (27 Candida glabrata, 10 C. tropicalis, 6 C. krusei, 6 C. parapsilosis, 4 C. lusitaniae, 4 C. lipolytica, etc.) were identified and tested. The antifungal susceptibility was estimated on the basis of minimum inhibitory concentrations (MIC). Results: Overall, 13 species were determined, of which C. glabrata was the most common (40.3%), followed by C. tropicalis (14.9%), C. krusei, and C. parapsilosis (8.9 % each). Forty-nine NAC isolates (73.13%) demonstrated decreased susceptibility to one or more antifungals, and 18 of them were resistant to all azoles. Out of 27 C. glabrata, 12 (44.4%) were resistant to fluconazole with MICs: 32 - >128 µg/mL and 15 (55.6%) were intermediate with MICs: 8 - 16 µg/mL Non-albicans Candida revealed a good susceptibility to echinocandins. Amphotericin B resistance was found in 5.97% of the isolates. Of particular interest was the detection of 6 (8.95%) multidrug-resistant NAC, which expressed resistance to azoles and echinocandins and/or amphotericin B. Conclusions: About one-fourth of the studied NAC were resistant to all azoles. These findings as well as the detection of several multidrug-resistant isolates determine the necessity of susceptibility testing of clinically important yeast isolates and control of the antifungal drugs in our hospital.

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Background Vancomycin-resistant enterococci (VRE) are well known agents that colonize the gastrointestinal tract of immunocompromised patients, especially those with hematologic malignancies. The aim of the current study was to determine the incidence of and risk factors for colonization with VRE among patients with hematologic malignancies. Materials For a nine-month period, all patients admitted to the Hematology ward at University Hospital in Pleven, Bulgaria who had hematologic malignancy and duration of hospitalization of more than 48 h were screened for colonization with VRE. The data collected from patients and their medical records during the entire hospital stay included: demographic characteristics, clinical information and information about all antimicrobials used. A longitudinal study was used to assesses the risk factors and statistical analysis was performed using SPSS version 27.0. Results A total of 119 patients were enrolled in the study. Colonization with VRE was established in 18 of them. One patient carried two species, resulting in a total of 19 VRE: 12 Enterococcus gallinarum, 4 Enterococcus casseliflavus, 2 Enterococcus faecium and 1 Enterococcus faecalis. VanA phenotype, with high-level resistance of vancomycin (MIC ≥ 256 μg/ml) and teicoplanin (MIC = 96 μg/ml), was demonstrated by one E. faecium, which carried vanA. The other E. faecium and E. faecalis expressed low-level resistance to vancomycin (MICs: 8 μg/ml and 12 μg/ml), susceptibility to teicoplanin (MICs = 0.5 μg/ml) and vanB was detected. All E. gallinarum and E. casseliflavus showed low-level resistance to vancomycin and susceptibility to teicoplanin. E. gallinarum strains were positive for vanC1 and E. casseliflavus for vanC2. Only two patients were colonized with vanA or vanB enterococci and the rest 16 were positive for vanC. The univariate analysis revealed that patient’s age (70–79 years; p = 0.025) and multiple myeloma (p = 0.001) are risk factors for VRE acquisition among the investigated patients. In addition, the multivariate analysis confirmed that patient’s age (70–79 years) is an independent risk factor for VRE colonization. Conclusions Our results showed that 15.1% of patients with hematologic malignancies were colonized with VRE. There was a distinct prevalence of vanC enterococci. Among the analyzed risk factors, advanced age and multiple myeloma contributed to VRE acquisition.