Molecular epidemiology and antimicrobial resistance of Haemophilus influenzae isolates from otitis media in Bulgaria.

Haemophilus influenzae is one of the main bacteria responsible for otitis media (OM) among children worldwide. We aimed to estimate the distribution of encapsulated and non-capsulated variants (NTHi), biotypes, antibiotic susceptibility, and molecular epidemiology of H. influenzae isolates recovered from pediatric OM cases in Bulgaria.Capsule detection was done by PCR for bexB gene, absent in NTHi. All encapsulated strains were subjected to PCR serotyping. MIC susceptibility testing was performed according to the criteria of EUCAST. MLST was conducted for all 71 OM isolates.The capsule detection and PCR - serotyping disclosed a predominance of NTHi (90.1%) and a few "a", "f", and "c" types. Biotype I was the most widespread (42.3%). ß-lactam resistance was found in 35.2% of the isolates. MLST represented heterogenic population structure, whereas the most represented clonal complexes belonged to ST-3, ST-57, ST-105, and ST-1426. 42.3% of the STs showed relatedness to globally represented clones, and 11.3% displayed affiliation to international type 2.Most of the H. influenzae isolates recovered from children with otitis media were non-typable strains from biotype I. The examined population structure was genetically diverse, with a predominance of international type 2 isolates.

Gut colonization with vancomicyn-resistant enterococci among patients with hematologic malignancies.

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.

Association of pili with widespread multidrug-resistant genetic lineages of non-invasive pediatric Streptococcus pneumoniae isolates.

The study aimed to evaluate the presence of pili in non-invasive pediatric pneumococcal isolates and to elucidate possible links with genetic lineages, serotypes, and antimicrobial resistance. We examined 147 Streptococcus pneumoniae isolates from children with respiratory tract infections and acute otitis media. Serotyping was performed by latex agglutination and capsule swelling reaction. Serogroup 6 was subjected to PCR-serotyping. Minimum inhibitory concentrations were determined according to EUCAST breakpoints. PCRs for rlrA and pitB genes were performed to detect a presence of type 1 and type 2 pili. MLST was conducted to define the clonal structure of the piliated strains. Almost all children (96.5%) were vaccinated with the pneumococcal conjugate vaccine PCV10. We detected 76.8% non-PCV10 - serotypes (NVTs) and 14.3% PCV10 serotypes. The predominant serotypes were NVTs: 19A (14.3%), 6C (12.2%), 3 (9.5%), 15A (7.5%) and 6A (6.8%). PI-1 was detected among 10.9% non-PCV10 serotypes 6A, 6C, and 19A and 6.1% PCV10 serotypes 19F and 23F. Type 2 pili were not found in the studied population. High levels of antimicrobial nonsusceptibility to erythromycin (58.5%), oral penicillin (55.8%), clindamycin (46.9%), trimethoprim-sulfamethoxazole (45.6%), tetracycline (39.5%) and ceftriaxone (16.3%) were revealed. The multidrug-resistant strains (MDR) were 55.1%. MLST represented 18 STs and three CCs among the piliated pneumococci: CC386, CC320, and CC81. More than half of the piliated strains (56.0%) belonged to successfully circulating international clones. PI-1 was associated mainly with MDR 6A, 6C, 19A, 19F, and 23F isolates from the widespread CC386, CC320, and CC81.

Clonal Distribution, Antimicrobial Resistance, and Pilus Islets in S. pneumoniae Isolates from PCV10-Vaccinated Children with Suppurative AOM in Bulgaria (2015‒2020).

Streptococcus pneumoniae is still a leading bacterial pathogen of acute otitis media (AOM), despite the availability of pneumococcal conjugate vaccines (PCVs). We conducted a study on the population structure, antibiotic nonsusceptibility, serotype distribution, and presence of pilus in middle ear fluids ‒ S. pneumoniae isolates recovered from PCV10-vaccinated children with suppurative АОМ in Bulgaria. Non-susceptibility was observed in 68.75% (n = 33) of the isolates, and multidrug resistance (MDR) was detected in 60.4% of the patients. The dual macrolide resistance mechanism was predominant. The most common serotypes were non-PCV10 serotypes 3 (27.1%, n = 13), 19A (25.0%, n = 12), and VT 19F (23.0%, n = 11). Overall, 64.6% were non-PCV10-serotypes. The presence of Pilus type I was observed mostly in the PCV10-serotypes. We found a strong association between clonal complexes (CCs), serotypes, and antimicrobial resistance. Multilocus sequence typing revealed the presence of four CCs: CC320 (39.6%), CC505 (12.5%), CC1377 8.3%), and CC230 (8.3%). The most abundant CC320 comprised MDR 19A and 19F isolates. CC230 clustered MDR isolates from serotypes 19A, 6C, and 14. CC505 and CC1377 were serotype 3 susceptible isolates. The vaccine-induced changes and trends in antimicrobial resistance and clonality must be the focus of systematic investigations.

Phenotypic and genotypic characterization of serogroup 6 Streptococcus pneumoniae isolates collected during 10-valent pneumococcal conjugate vaccine era in Bulgaria.

Serogroup 6 remains common in the pneumococcal-conjugated vaccine era in Bulgaria; therefore, we investigated its clonal and serotype dynamics. The antibiotic susceptibilities were assessed by broth microdilution. Strains identified as serogroup 6 with latex agglutination method were subjected to serotype-specific PCRs. Erythromycin-resistant strains were analyzed by PCR for presence of ermB and mefE genes. MLST was performed to define clonal composition of the sequence types (STs). Serogroup 6 was represented by 40 (13.3%) from 301 invasive and non-invasive Streptococcus pneumoniae isolates. Molecular serotyping revealed new emerging serotype 6C (6.6%), not detected in pre-vaccine era. Among unvaccinated patients, mostly we observed serotypes 6А (57.1%) and 6В (28.6%). Serotype 6C was distinctive for vaccinated children (64%), followed by 6A (24%). Penicillin and ceftriaxone non-susceptible serogroup 6 strains were 65% and 5%, respectively; erythromycin- and clindamycin-resistant were 70.0% and 52.5%, respectively. Multidrug-resistant strains were 57.5%. Prevalent genetic determinant for macrolide resistance was ermB gene (75%). MLST revealed 17 STs into 5 clonal complexes and 7 singletons. Predominant genetic lineage was CC386, represented by MDR-6C non-invasive strains. Serotype 6B, principally responsible for invasive diseases in the pre-vaccine era, retreated this position to serotype 6A.