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.

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.

Addition of Medicinal Plants Increases Antioxidant Activity, Color, and Anthocyanin Stability of Black Chokeberry (Aronia melanocarpa) Functional Beverages.

The present study investigates the effect of the addition of medicinal plants, such as lady's mantle, lavender, rosehip, and meadowsweet, on the chemical composition, antioxidant activity, and color intensity of ready-to-drink aronia nectar during pasteurization and long-term storage. Pasteurization caused a significant decrease in anthocyanin content of aronia nectar, which reduced to 20% of the initial value after four months of storage. Herbs provided different protection to aronia anthocyanins that degraded more slowly during the four-month storage compared to pasteurized control without herbs. The addition of medicinal plants enriched aronia nectar with phenolic compounds and increased its antioxidant activity by up to 52% in meadowsweet-aronia nectar. Moreover, it was accompanied by a color intensity magnification due to co-pigmentation of aronia anthocyanins and herbal phenolics. In contrast to anthocyanins, which constantly degraded during the whole period, color intensity began to stabilize after 30 days, demonstrating that co-pigmentation was progressively established during the time and rosehip provided the best stabilization of aronia nectar color. Current research demonstrates for the first time that medicinal plants such as lady's mantle, rosehip, and especially meadowsweet can be used to increase antioxidant activity, color, and anthocyanin stability of black chokeberry functional beverages.

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.

Validation of HPLC Method for Analysis of Gamma-Aminobutyric and Glutamic Acids in Plant Foods and Medicinal Plants.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system of mammals and plays an important role in the suppression of neurons' excitability. GABA is formed from the decarboxylation of glutamic acid (Glu), and both GABA and Glu could be considered as important biologically active food components. In the current study, we validated a HPLC method for concomitant detection of GABA and Glu in plant samples after derivatization with dansyl chloride. The validated method had high precision and a high recovery rate and was successfully used for GABA and Glu quantification in 55 plant foods (fruits, vegetables, legumes, cereals, pseudocereals, and nuts) and 19 medicinal plants. Vegetables were the most important dietary source of these amino acids, with the highest quantity of GABA found in potatoes-44.86 mg/100 g fresh weight (FW) and yellow cherry tomatoes-36.82 mg/100 g FW. The highest amount of Glu (53.58 mg/100 g FW) was found in red cherry tomatoes. Analyzed fruits were relatively poor in GABA and Glu, and European gooseberry was the richest fruit with 13.18 mg/100 g FW GABA and 10.95 mg/100 g FW Glu. Cereals, pseudocereals, nuts, and legumes contain much higher amounts of Glu than GABA. The obtained results enrich the available information on the content of gamma-aminobutyric and glutamic acids in plant foods and could be used for the development of GABA-enriched functional foods.

Fecal Carriage and Epidemiology of Extended-Spectrum Beta-Lactamase/Carbapenemases Producing Enterobacterales Isolates in Bulgarian Hospitals.

The gastrointestinal tract is an important reservoir of extended spectrum beta-lactamase (ESBL)/carbapenemase-producing Enterobacterales isolates. This study included patients from two Bulgarian hospitals. Overall, 98 ESBL producers (including 68 Escherichia coli and 20 Klebsiella pneumoniae isolates) were detected among 99 hospitalized patients, 212 patients at admission, and 92 hospital staff in 42.4%, 24.5%, and 4%, respectively. We observed blaCTX-M-15 in 47% of isolates, blaCTX-M-3 in 39% and blaCTX-M-14 in 11%. Three blaCTX-M-15 positive isolates were also blaKPC-2 positive. High transferability was detected for blaCTX-M-3 carrying plasmids (55%) with L/M and I1 replicon plasmids, followed by CTX-M-14 (36.4%) and CTX-M-15 (27.9%) with IncF plasmids. BlaKPC-2 was carried by FIIAs plasmids. Epidemiology typing revealed 8 K. pneumoniae ST types-ST15(8/20), ST17(4/20), ST37(2/20) and 9 E. coli ST types-ST131 (30.9%, 21/68), ST38 (8/68), ST95(7/68) and ST316(7/68). All ST131 isolates but one was from the highly virulent epidemic clone O25bST131. This is the first report in Bulgaria about ESBL/carbapenemase faecal carriage. We observed high ESBL/carbapenemases prevalence. A predominant number of isolates were members of highly epidemic and virulent PanEuropean clones ST15 K. pneumoniae and O25bST131 E. coli. High antibiotics usage during the COVID pandemic will worsen the situation. Routine screenings and strict infection control measures should be widely implemented.

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.

Dissemination of successful international clone ST15 and clonal complex 17 among Bulgarian CTX-M-15 producing K. pneumoniae isolates.

A total of 82 extended spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae and 4 Klebsiella oxytoca isolates were collected in 2014 from four geographical areas in Bulgaria and their multilocus sequence type (MLST) and transferability of the ESBL encoding genes were investigated. The predominant type was CTX-M-15 (87%), followed by CTX-M-3 (9%), SHV-12 or SHV-2 (2%) and CTX-M-14 (1%). The CTX-M-15 producers belonged to ST15 (34.1%) and to a lesser extent to CC17 (ST16, ST17, ST336). The CTX-M-15 transconjugants showed a presence of R, A/C2 and F replicons. The CTX-M-3 producers were assigned to ST29, ST70, ST432, ST542 and ST15 types and the transconjugants carried M2 replicons. To the best of our knowledge, this is the first report that fully describes the MLST types among Bulgarian ESBL producing K. pneumoniae and the first report of the detection of IncR plasmid replicon type in our country.

Sporocidic properties of poly(vinyl alcohol)/silver nanoparticles/TEOS thin hybrid films.

The sporocidic activity of hybrid materials based on PVA/AgNps/TEOS thin films has been investigated. Deep Agar Method has been applied to study the sporocidic properties of these hybrid materials with different silver concentrations. This method has been used because of the lack of standard methods for testing the sporocidic activity in such materials and due to the specific characteristics of bacterial spore. Clear and pronounced presence of sporocidic activity of the hybrid materials towards spores of control strains Bacillus subtilis ATCC 6633 and Geobacillus stearothermophilus ATCC 7953 has been established. The use of chromatographic paper disks impregnated with PVA/AgNps/TEOS showed the advantages in testing the biological properties of the hybrid material in comparison to the disks obtained by directly cutting the PVA/AgNps/TEOS films. The highest sporocidic activity, although with small deviation of 0.5-1.0 mm, was established at the PVA/AgNps/TEOS hybrid films with concentration of silver precursor 9.2 mg/mL and 18.3 mg/mL. The experiments were performed with the aim to reveal the opportunities for a practical application of the material.

Polyvinyl alcohol/silver nanoparticles (PVA/AgNps) as a model for testing the biological activity of hybrid materials with included silver nanoparticles.

Comparing the influence of two different stabilizers as polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), as well as organosilanes as tetraethoxysilane (TEOS), allows determining the main role of the silver nanoparticles included in hybrid materials for the realization of their antimicrobial activity. The proposed two-step testing scheme first onto control strains and then onto clinical bacterial and fungal strains resistant to antibiotics allows full investigation of these properties.

Synthesis and comparative study on the antimicrobial activity of hybrid materials based on silver nanoparticles (AgNps) stabilized by polyvinylpyrrolidone (PVP).

Hybrid materials based on polyvinylpyrrolidone (PVP) with silver nanoparticles (AgNps) were synthesized applying two different strategies based on thermal or chemical reduction of silver ions to silver nanoparticles using PVP as a stabilizer. The formation of spherical silver nanoparticles with diameter ranging from 9 to 16 nm was confirmed by TEM analysis. UV-vis and FTIR spectroscopy were also applied to confirm the successful formation of AgNps. The antibacterial activity of the synthesized AgNPs/PVP against etalon strains of three different groups of bacteria-Staphylococcus aureus (S. aureus; gram-positive bacteria), Escherichia coli (E. coli; gram-negative bacteria), Pseudomonas aeruginosa (P. aeruginosa; non-ferment gram-negative bacteria), as well as against spores of Bacillus subtilis (B. subtilis) was studied. AgNps/PVP were tested for the presence of fungicidal activity against different yeasts and mold such as Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Aspergillus brasiliensis. The hybrid materials showed a strong antimicrobial effect against the tested bacterial and fungal strains and therefore have potential applications in biotechnology and biomedical science.

Synthesis, characterisation and antibacterial activity of PVA/TEOS/Ag-Np hybrid thin films.

Novel hybrid material thin films based on polyvinyl alcohol (PVA)/tetraethyl orthosilicate (TEOS) with embedded silver nanoparticles (AgNps) were synthesized using sol-gel method. Two different strategies for the synthesis of silver nanoparticles in PVA/TEOS matrix were applied based on reduction of the silver ions by thermal annealing of the films or by preliminary preparation of silver nanoparticles using PVA as a reducing agent. The successful incorporation of silver nanoparticles ranging from 5 to 7nm in PVA/TEOS matrix was confirmed by TEM and EDX analysis, UV-Vis spectroscopy and XRD analysis. The antibacterial activity of the synthesized hybrid materials against etalon strains of three different groups of bacteria -Staphylococcus aureus (gram-positive bacteria), Escherichia coli (gram-negative bacteria), Pseudomonas aeruginosa (non-ferment gram-negative bacteria) has been studied as they are commonly found in hospital environment. The hybrid materials showed a strong bactericidal effect against E. coli, S. aureus and P. aeruginosa and therefore have potential applications in biotechnology and biomedical science.

Antibacterial activity of poly(vinyl alcohol)-b-poly(acrylonitrile) based micelles loaded with silver nanoparticles.

A new amphiphilic poly(vinyl alcohol)-b-poly(acrylonitrile) (PVOH-b-PAN) copolymer obtained by selective hydrolysis of well-defined poly(vinyl acetate)-b-poly(acrylonitrile) copolymer synthesized by cobalt mediated radical polymerization was used for the preparation of PVOH-b-PAN based micelles with embedded silver nanoparticles. The successful formation of silver loaded micelles has been confirmed by UV-vis, DLS and TEM analysis and their antibacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and spore solution of Bacillus subtilis (B. subtilis) has been studied. PVOH-b-PAN based micelles with embedded silver nanoparticles showed a strong bactericidal effect against E. coli, S. aureus and P. aeruginosa and the minimum bactericidal concentration for each system (MBC) has been determined.