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.

Molecular epidemiology, virulence and antimicrobial resistance of Bulgarian methicillin resistant Staphylococcus aureus isolates.

Background: Severe infections of virulent methicillin-resistant Staphylococcus aureus (MRSA) are a serious health problem. The present study aimed to investigate clonal spread, virulence and antimicrobial resistance rates of Bulgarian MRSA isolates in 2016-2020. Methods: Molecular identification and mecA gene detection were performed with PCR. Clonal relatedness was evaluated by RAPD PCR and MLST. MRSA epidemiology, virulence and resistance patterns were investigated by PCR. Results: All 27 isolates were identified as S. aureus and were mecA positive, and all were susceptible to linezolid, tigecycline and vancomycin. The toxin genes hlg (in 92.6% of isolates), seb (77.8%), sei (77.8%), seh (59.3%), sej (55.6%), and seg (48.1%), were frequently found among the isolates. Epidemiological typing by RAPD identified 4 clones (16 isolates) and 11 were with a unique profile. MLST analysis of the same MRSA isolates showed five MLST clonal complexes and 11 ST types, including CC5 (33.3%) (ST5, ST221, ST4776), CC8 (22.2%) (ST8, ST239, ST72), CC15 (ST582), CC22 (14.8%) (ST217, ST5417), CC30 (ST30) CC398 (ST398), and CC59 (ST59). The isolates from CC5 showed higher virulence potential and almost all were macrolide resistant (ermB or ermC positive). CC8 isolates showed higher level of resistance. Conclusion: To the best of our knowledge, this study is the first describing the clonal spreading of Bulgarian MRSA and the association with their virulence and resistance determinants. Monitoring of MRSA epidemiology, resistance and virulence profile can lead to better prevention and faster therapeutic choice in cases of severe infections.

The role of IL-17 rs2275913, IL-17RC rs708567 and TGFB1 rs1800469 SNPs and IL-17A serum levels in patients with lupus nephritis.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease and polymorphisms in the cytokine genes and their receptors are thought to influence its development. The aim of this case-control study was to investigate the association of the IL-17A rs2275913, IL-17RC rs708567 and TGFB1 rs1800469 polymorphisms with SLE, its clinical manifestations and the polymorphisms influence on the IL-17A serum levels. Altogether 59 SLE patients with lupus nephritis and 95 healthy controls were genotyped by TaqMan assay. Serum levels were determined by Human IL-17A Platinum ELISA kit. From the studied polymorphisms, only TGFB1 T allele was found to be associated with SLE. Within the patient group, IL-17A GG genotype and TGFB1 -509T allele showed an association with the neurological disease and IL-17RC CC genotype appeared to be associated with lupus arthritis. The IL17A serum levels in the SLE and control groups (7.24 pg/ml and 5.76 pg/ml, respectively) did not show any statistical difference. A weak correlation between IL17A levels and SLEDAI-2K was observed. Our results indicate that IL-17A rs2275913, IL-17RCrs708567 and TGFB1 rs1800469 polymorphisms might play a role in the susceptibility and the clinical manifestations of SLE and IL-17A serum levels should be monitored in the course of the disease. The identification of subsets of SLE with an IL-17-driven disease could improve the therapeutic approach leading to more precise personalized treatment.

Molecular emm typing of Bulgarian macrolide-resistant Streptococcus pyogenes isolates.

Group A streptococcus (GAS) is a human pathogen causing a broad range of infections, linked with global morbidity and mortality. Macrolide resistance rates vary significantly in different parts of the world. Driving factors of the emergence and spread of resistant clones are not clearly understood. We investigated 102 macrolide-resistant GAS strains collected during the period 2014-2018 from various clinical specimens from Bulgarian patients. Strains were characterized by the presence of mefA/mefE, ermA, and ermB using polymerase chain reaction and sequencing for mefA/mefE. Resistant strains were studied by emm sequence typing and emm-cluster system. Most prevalent emm types among the macrolide-resistant GAS strains were emm28 (22.55%), emm12 (17.65%), and emm4 (16.66%). Almost all (87.25%) of the macrolide-resistant isolates harboring ermB were emm28. The isolates that carried ermA were predominantly emm12 (38.24%) and emm77 (38.24%), with fewer emm89 (23.53%). The isolates harbored predominantly mefE (49 isolates) and only 9 strains carried mefA. The most prevalent emm clusters among the GAS isolates were E4 (40.20%), A-C4 (17.65%), and E1 (16.66%). The study's results suggest that dissemination of specific clones in GAS population may also be the reason for the increasing macrolide-resistance rate in our country.

In Vitro Antimicrobial Activity of Five Newly Approved Antibiotics against Carbapenemase-Producing Enterobacteria-A Pilot Study in Bulgaria.

To solve the problem with pan-drug resistant and extensively drug-resistant Gram-negative microbes, newly approved drugs such as ceftazidime/avibactam, cefiderocol, plazomicin, meropenem/vaborbactam, and eravacycline have been introduced in practice. The aim of the present study was to collect carbapenemase-producing clinical Enterobacterales isolates, to characterize their carbapenemase genes and clonal relatedness, and to detect their susceptibility to commonly used antimicrobials and the above-mentioned newly approved antibiotics. Sixty-four carbapenemase producers were collected in a period of one year from four Bulgarian hospitals, mainly including Klebsiella pneumoniae (89% of the isolates) and also single Proteus mirabilis, Providencia stuartii and Citrobacter freundii isolates. The main genotype was blaNDM-1 (in 61%), followed by blaKPC-2 (23%), blaVIM-1 (7.8%) and blaOXA-48 (7.8%). Many isolates showed the presence of ESBL (blaCTX-M-15/-3 in 76.6%) and AmpC (blaCMY-4 in 37.5% or blaCMY-99 in 7.8% of isolates). The most common MLST type was K. pneumoniae ST11 (57.8%), followed by ST340 (12.5%), ST258 (6.3%) and ST101 (6.3%). The isolates were highly resistant to standard-group antibiotics, except they were susceptible to tigecycline (83.1%), colistin (79.7%), fosfomycin (32.8%), and aminoglycosides (20.3-35.9%). Among the newly approved compounds, plazomicin (90.6%) and eravacycline (76.3%) showed the best activity. Susceptibility to ceftazidime/avibactam and meropenem/vaborbactam was 34.4% and 27.6%, respectively. For cefiderocol, a large discrepancy was observed between the percentages of susceptible isolates according to EUCAST susceptibility breakpoints (37.5%) and those of CLSI (71.8%), detected by the disk diffusion method. This study is the first report to show patterns of susceptibility to five newly approved antibiotics among molecularly characterized isolates in Bulgaria. The data may contribute to both the improvement of treatment of individual patients and the choice of infection control strategy and antibiotic policy.

Introducing exome sequencing as part of the diagnostic algorithm for pediatric nephrology patients in Bulgaria - a single-center experience.

INTRODUCTION: In pediatric kidney patients, where clinical presentation is often not fully developed and renal biopsy too risky or inconclusive, it may be difficult to establish the underlying pathology. In cases such as these, genetic diagnosis may be used to guide the treatment, prognosis and counselling. Given the large number of genes involved in kidney disease, introducing next generation sequencing with extended gene panels as part of the diagnostic algorithm presents a viable solution. METHODS: A cohort of 87 consecutive independent cases (83 children and 4 terminated pregnancies) with renal disease were recruited. Exome sequencing with MiSeq or NovaSeq 6 000 (Illumina) platforms and analysis of extended gene panels was used for genetic testing. RESULTS: Depending on the presenting pathology, the cases were grouped as patients with glomerular disease, ciliopathies, congenital anomalies, renal electrolyte imbalances and chronic/acute kidney disease. The overall diagnostic yield was app. 42% (37 out of 87) with most disease-causing mutations found in COL4A3, COL4A4, COL4A5 and PKHD1 genes. A change or clarification of preliminary diagnosis, or adjustment of initial treatment plan based on the results from the genetic testing was made for app. one third of the children with meaningful genetic findings (11 out 37). DISCUSSION: Our results prove the value of targeted exome sequencing as non-invasive, versatile and reliable diagnostic tool for pediatric renal disease patients. Providing genetic diagnosis will help for better understanding of disease etiology and will give basis for optimal clinical management and insightful genetic counseling.

Development of Nested PCR for SARS-CoV-2 Detection and Its Application for Diagnosis of Active Infection in Cats.

SARS-CoV-2 emerged in 2019 and found diagnostic laboratories unprepared worldwide. To meet the need for timely and accurate virus detection, laboratories used rapid Ag tests and PCR kits based on costly multi-channel real-time techniques. This study aimed to develop a conventional nested PCR based on the SARS-CoV-2 N gene, validate it against some approved assays, and apply it to samples from six cats with respiratory symptoms obtained in early 2020 during the first COVID-19 wave in humans in Bulgaria. The nested PCR technique showed 100% sensitivity and specificity; it could detect extracted SARS-CoV-2 RNA at concentrations as low as 0.015 ng/µL. The results identified the six tested cat samples as positive. Sequence analysis performed in two of them confirmed this. The presented technique is reliable, easy to implement and inexpensive, and can be successful in strategies for the prevention and control of SARS-CoV-2 in humans, cats and other susceptible species.

Panel-based next-generation sequencing identifies novel mutations in Bulgarian patients with inherited retinal dystrophies.

BACKGROUND: Next-generation sequencing (NGS)-based method is being used broadly for genetic testing especially for clinically and genetically heterogeneous disorders, such as inherited retinal degenerations (IRDs) but still not routinely used for molecular diagnostics in Bulgaria. Consequently, the purpose of this study was to evaluate the effectiveness of a molecular diagnostic approach, based on targeted NGS for the identification of the disease-causing mutations in 16 Bulgarian patients with different IRDs. METHODS: We applied a customized NGS panel, including 125 genes associated with retinal and other eye diseases to the patients with hereditary retinopathies. RESULTS: Systematic filtering approach coupled with copy number variation analysis and segregation study lead to the identification of 16 pathogenic and likely pathogenic variants in 12/16 (75%) of IRD patients, 2 of which novel (12.5%): ABCA4-c.668delA (p.K223Rfs18) and RР1-c.2015dupA (p.K673Efs*25). Mutations in the ABCA4, PRPH2, USH2A, BEST1, RР1, CDHR1, and RHO genes were detected reaching a diagnostic yield between 42.9% for Retinitis pigmentosa cases and 100% for macular degeneration, Usher syndrome, and cone-rod dystrophy patients. CONCLUSION: Our results confirm the usefulness of targeted NGS approach based on frequently mutated genes as a comprehensive and successful genetic diagnostic tool for IRDs with significant impact on patients counseling.

Dissemination of High-Risk Clones Enterobacterales among Bulgarian Fecal Carriage Isolates.

The gastrointestinal tract is an important reservoir of high-risk Enterobacteria clones and a driver of antimicrobial resistance in hospitals. In this study, patients from six hospitals in four major Bulgarian towns were included in this study. Overall, 205 cefotaxime-resistant isolates (35.3%) of Enterobacterales order were detected in fecal samples among 580 patients during the period of 2017-2019. ESBL/carbapenemase/plasmidic AmpC producer rates were 28.8%, 2.4%, and 1.2%, respectively. A wide variety of ESBLs: CTX-M-15 (41%), CTX-M-3 (24%), CTX-M-27 (11%), and CTX-M-14 (4%) was found. The carbapenemases identified in this study were New Delhi metalo-ß-lactamase (NDM)-1 (5.4%) and Klebsiella carbapenemase (KPC)-2 (1.5%). Most NDM-1 isolates also produced CTX-M-15/-3 and CMY-4 ß-lactamases. They belonged to ST11 Klebsiella pneumoniae clone. The epidemiology typing revealed three main high-risk K. pneumoniae clones (26%)-ST11, ST258, and ST15 and five main Escherichia coli clones-ST131 (41.7%), ST38, ST95, ST405, and ST69. Sixty-one percent of ST131 isolates were from the highly virulent epidemic clone O25b:H4-ST131. Phylotyping revealed that 69% of E. coli isolates belonged to the virulent B2 and D groups. Almost all (15/16) Enterobacter isolates were identified as E. hormaechei and the most common ST type was ST90. Among all of the isolates, a high ESBL/carbapenemases/plasmid AmpC (32.4%) prevalence was observed. A significant proportion of the isolates (37%) were members of high-risk clones including two pan-drug-resistant K. pneumoniae ST11 NDM-1 producing isolates. Due to extensive antibiotic usage during COVID-19, the situation may worsen, so routine screenings and strict infection control measures should be widely implemented.

A rare case of RGR/CDHR1 haplotype identified in Bulgarian patient with cone-rod dystrophy.

AIM: To present a rare clinical case of CDHR1-related retinopathy with cone and rod involvementconfirmed clinically, electrophysiologically and genetically as a cone-rod dystrophy. MATERIAL AND METHODS: A 26-year-old woman underwent detailed ophthalmic examinationincluding fundus photography, full-field and multifocal electroretinography, visual field testing, optical coherence tomography and fluorescein angiography, which established the clinical diagnosis. Next-generation sequencing of a custom panel including 140 of the most common genes for inherited retinal degenerations was used for mutation screening. RESULTS: The symptoms onset was two years ago included gradual loss of vision and photophobia. The clinical findings were reduced visual acuity, central and peripheral scotomas, sporadic pigmentary cells localized mainly in the peripheral retina, a thinner retina in the macula and peripherally, moderate retinal vessels attenuation and reduced cone and rod ERG responses. The genetic analysisfound that the patient was homozygous for two already reported mutations: RGR-c.196A>C (p.Ser66Arg) variant and a co-segregating frame-shift deletion in CDHR1-c.2522_2528delTCTCTGA (p.Ile841Serfs119*). Segregation analysis showed that the two mutations were transmitted by the asymptomatic heterozygous parents. CONCLUSION: The rare haplotype of RGR mutation co-segregating incis- with CDHR1 mutation in our patient has been previously described in Albanian patients with recessive retinal dystrophy. Our findings add further support to the hypothesis of a common ancestral haplotype spread in the Balkan population. The comprehensive clinical, electrophysiological and genetic testing of patients with rare hereditary retinal dystrophies is essential for the correct diagnosis and the choice of potential novel therapies.

Relation between emm types and virulence gene profiles among Bulgarian Streptococcus pyogenes clinical isolates.

Background: The Streptococcus pyogenes emm gene, which encodes M protein, is an important epidemiological marker. The aim of this study is to determine the emm genotypes of Bulgarian clinical streptococccal isolates in 2014-2018 and to evaluate their relationship with virulence genes profiling and disease types. Methods: PCR and sequencing were used for emm genotyping of 182 S. pyogenes clinical isolates according to the protocol of the Centre for Disease Control and Prevention. PCR was used to investigate the virulence factors. Results: We identified 15 emm types and eight clusters. Five main clusters with eight emm types were predominant: cluster A-C3 (emm1) - 24.7%, A-C5 (emm3) - 19.2%, E1 (emm4) - 11.0%, A-C4 (emm12) - 11.0% and E4 (emm2,28,77,89) - 20.9%. There were two novel subtypes: emm3.132 and emm3.133. The investigated strains with emm3 genotypes were common in sterile site infections (invasive ones) and types emm4 and emm12, in skin and mucosal infections. More than 60% of the major cluster A-C3 (emm1; emm1.33; emm1.6) members possessed many genes for streptococcal pyrogenic exotoxins that act as super-antigens and bring about potentially higher virulence. Conclusion: The present study described two novel emm3 subtypes. To the best of our knowledge, this study is the first that describe the emm type spectrum of Bulgarian S. pyogenes clinical isolates and associated virulence factors. Monitoring of the S. pyogenes pathogenic potential and epidemiology can lead to better knowledge and higher possibility for prevention and eradication of complications of streptococcal infections.

Quinolone resistance mechanisms among third-generation cephalosporin resistant isolates of Enterobacter spp. in a Bulgarian university hospital.

Background: There have been no reports in Bulgaria about quinolone resistance determinants among Enterobacter spp. Aims: To investigate plasmid and chromosomal quinolone resistance rates among 175 third-generation cephalosporin resistant Enterobacter spp. isolates (167 Enterobacter cloacae complex and eight Enterobacter aerogenes isolates) collected at a university hospital in Varna, Bulgaria, as well as to reveal their association with ESBL/AmpC production and a carriage of specific plasmid replicon types. Methods: PCR, isoelectric focusing, replicon typing, sequencing, and epidemiology typing were carried out. Results: A high level of combined third-generation cephalosporin and quinolone resistant Enterobacter spp. was found - 79.4%. The ESBL production rate was 87%, consisting mainly of CTX-M-15 among E. cloacae complex (in 76%) and CTX-M-3 among E. aerogenes (in 88%). Plasmid mediated quinolone resistance (PMQR) determinants were identified in 57% of the isolates. The most commonly detected PMQR determinants were qnrB (90%), consisting mainly of qnrB1 (in 61%), and qnrB9 (in 27%) of the isolates. Both alleles were transferred with CTX-M-15 genes; transconjugants showed HI2 replicons (for qnrB1 positive transconjugants) and were non-typeable (for qnrB9). One Enterobacter spp. isolate produced qnrB4. QnrA1, qnrS1, and aac(6')-Ib-cr were detected in single isolates only. QnrC, qnrD, qepA, and oqxAB genes were not found. QnrB was associated with CTX-M-15 production, and qnrS1 was linked to CTX-M-3. Alterations in 83 and 87 positions of gyrB in quinolone-resistance determining regions, and 80 position of parC were detected in high level quinolone resistant isolates. Among all the Enterobacter spp. isolates tested, one predominant clone A was identified (53%). Conclusion: Our data showed the necessity of more prudent use of quinolones and third-generation cephalosporins, because of the risk of promoting dissemination, and selection of multiple resistance determinants (ESBL, PMQR) among Enterobacter spp. isolates in Bulgaria.

First Report of DHA-1 Producing Enterobacter cloacae Complex Isolate in Bulgaria.

THE AIM: of the present study was to reveal the characteristics of an. MATERIALS AND METHODS: Susceptibility testing, conjugation experiments, isoelectric focusing, PCR and sequencing were carrying out. RESULTS: Of 176. CONCLUSIONS: To the best of our knowledge this is the first report of DHA-1 producing isolate in Bulgaria. The emergence of DHA-1 producing.

Multicentre investigation of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in Bulgarian hospitals - Interregional spread of ST11 NDM-1-producing K. pneumoniae.

AIM: The aim of this study was to investigate the mechanisms of beta-lactam-resistance and the clonal relatedness of carbapenem-nonsusceptible Klebsiella pneumoniae and Escherichia coli isolates, collected consecutively in eight centers in five Bulgarian cities from November 2014 to March 2018. Carbapenemase-producing enterobacteria were detected in all but one centers. Overall, 104 K. pneumoniae and one E. coli were analysed. MATERIALS AND METHODS: Antimicrobial susceptibility and beta-lactamases were analysed. Conjugation experiments, plasmid fingerprinting and replicon typing, as well as MLST and ERIC-PCR were carried out. RESULTS: KPC-2 (51%) and NDM-1 (47%) were the main carbapenemases identified. KPC-2 producing K. pneumoniae were classified into 10 MLST-types. The four dominating MLST-types ST29, ST15, ST336 and ST902 comprised 79% of the KPC-2 producers. All but one of the NDM-1 producing isolates belonged to the MLST-type ST11 and were found in seven centers. Furthermore, single K. pneumoniae isolates producing VIM-1 (ST147) and OXA-48 (ST15) were identified. In addition to the carbapenemases, the ESBLs CTX-M-15, CTX-M-3, and SHV-12 as well as AmpC enzyme CMY-4 were found. The FIIAs-replicon-type was found in all KPC-2 producers while the A/C-replicons dominated in NDM-1 producing isolates. The single NDM-1 producing E. coli was determined as MLST-Type ST10 (Warwick scheme). CONCLUSION: The interregional clonal expansion of NDM-1 producing ST11 K. pneumoniae and the dissemination of blaKPC-2 carrying plasmids were responsible for the spread of carbapenemase-producing K. pneumoniae in Bulgaria. Our findings highlight the urgency to prevent dissemination of these highly transmissible and dangerous lineages.

Cognitive, neuropsychiatric, and motor features associated with apolipoprotein E ε4 allele in a sample of Bulgarian patients with late-onset Parkinson's disease.

OBJECTIVE: To evaluate the role of apolipoprotein E (APOE) ε4 allele on cognitive, neuropsychiatric, and motor features in a sample of Bulgarian patients with late-onset Parkinson's disease (LOPD, age at onset > 55 years). METHODS: A total of 16 patients with LOPD having APOE ε3/ε4 genotype were compared to 30 patients with LOPD having APOE ε3/ε3 genotype and 20 healthy control individuals. Detailed cognitive assessment and evaluation of neuropsychiatric and motor symptoms were performed. RESULTS: The patients with LOPD had significantly lower scores in all cognitive domains compared to controls. The patients with LOPD carrying an ε4 allele showed some significant differences in their cognitive, motor, and neuropsychiatric features. CONCLUSIONS: The data suggest a role of the APOE genotype as a disease-modifying factor.