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The present study aimed to explore the genotypic and phenotypic characteristics of biofilm formation in Bulgarian nosocomial Stenotrophomonas maltophilia isolates (n = 221) during the period 2011-2022, by screening for the presence of biofilm-associated genes (BAG) (spgM, rmlA and rpfF), their mutational variability, and assessment of the adherent growth on a polystyrene surface. The methodology included: PCR amplification, whole-genome sequencing (WGS) and crystal violet microtiter plate assay for biofilm quantification. The overall incidence of BAG was: spgM 98.6%, rmlA 86%, and rpfF 66.5%. The most prevalent genotype was spgM+/rmlA+/rpfF+ (56.1%), followed by spgM+/rmlA+/rpfF- (28.5%), and spgM+/rmlA-/rpfF+ (9.5%), with their significant predominance in lower respiratory tract isolates compared to those with other origin (P < 0.001). All strains examined were characterized as strong biofilm producers (OD550 from 0.224 ± 0.049 to 2.065 ± 0.023) with a single exception that showed a weak biofilm-forming ability (0.177 ± 0.024). No significant differences were observed in the biofilm formation according to the isolation source, as well as among COVID-19 and non-COVID-19 isolates (1.256 ± 0.028 vs. 1.348 ± 0.128, respectively). Also, no correlation was found between the biofilm amounts and the corresponding genotypes. WGS showed that the rmlA accumulated a larger number of variants (0.0086 per base) compared to the other BAG, suggesting no critical role of its product to the biofilm formation. Additionally, two of the isolates were found to harbour class 1 integrons (7-kb and 2.6-kb sized, respectively) containing sul1 in their 3' conservative ends, which confers sulfonamide resistance. To the best of our knowledge, this is the first study on S. maltophilia biofilm formation in Bulgaria, which also identifies novel sequence types (ST819, ST820 and ST826). It demonstrates the complex nature of this adaptive mechanism in the multifactorial pathogenesis of biofilm-associated infections.

Purpose: The non-invasive pneumococcal disease (NIPD) is a common infection during childhood. We aimed to define the clonal spread of pediatric non-invasive isolates recovered during the PCV10-period in Bulgaria concerning the serotype and antimicrobial susceptibility., Materials and Methods: Serogrouping/serotyping were performed using latex agglutination and capsular swelling reaction. Serogroup 6 strains were subjected to serotype-specific PCR's. The antibiotic susceptibilities were assessed by broth microdilution. MLST was performed to define the clonal composition., Results: We analyzed 154 pediatrics non-invasive S. pneumoniae isolates. The PCV10-vaccinated children were 94.1%. We disclosed 88% non-vaccine serotypes (NVTs) and 12% PCV10 - serotypes. All common serotypes among PCV10-vaccinated children (n ​= ​145) were non-vaccine types (NVTs): 19A (13.8%), 6C (11.7%), 3 (9.6%), 15A (8.3%) and 23A (5.5%). Antimicrobial non-susceptibility showed highest levels in erythromycin (50.0%), oral penicillin (49.4%), clindamycin (45.4%), trimethoprim-sulfamethoxazole (43.5%), tetracycline (42.2%), and ceftriaxone (14.3%). The multidrug-resistant strains (MDR) were 51.3%. MDR-serotypes were 6C (20.2%), 19A (17.7%), 15A (11.4%), 19F (10.1%), and 23A (8.9%). MLST presented 17 clonal complexes (CCs) with prevalence of CC320, CC386, CC505, CC8029 and CC2613 clustered 83% MDR isolates., Conclusions: All emergent pediatric non-invasive serotypes in our geographic area during the studied PCV10-period were NVTs (19A, 6C, 3, 15A, and 23A). The fifth widespread CCs: CC320, CC386, CC505, CC8029 and CC2613 clustered 83% MDR isolates. Future surveillance of vaccine-induced changes in the clonality and the antimicrobial resistance of the pneumococcal population is needed., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

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 emm 3 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.

Objectives: Pneumococcal conjugate vaccines (PCVs) have reduced the incidence of pneumococcal disease, but non-vaccine serotypes are of concern, particularly if antimicrobial-resistant. This study retrospectively evaluated the serotype-specific clonality of paediatric multidrug-resistant (MDR) invasive and non-invasive Streptococcus pneumoniae isolates collected following PCV10 introduction (2011-2017) in Bulgaria., Methods: Capsular types, drug resistance patterns and multilocus sequence typing (MLST) of the most common MDR S. pneumoniae serotypes sampled from children were determined., Results: Overall, the rate of MDR pneumococci was 44.6% (107/240). The most common serotypes among MDR strains were 19F (25.2%), 19A (19.6%), 6C (13.1%), 6A and 23A (6.5% each) and 15A (4.7%), contributing 75.7% of all MDR strains. With the exception of serotype 19F, the remaining serotypes were non-PCV10 types. Among MDR pneumococci, the most frequently found sequence types were ST320 (30.4%; 19A and 19F), ST386 (12.7%; 6C and 6A) and ST8029 (5.1%; 23A). The majority of MDR STs (74.7%) belonged to PMEN clonal complexes, of which the most common were CC320 (Taiwan 19F -14), CC315 (Poland 6B -20) and CC180 (Netherlands 3 -31), accounting for 43.0%, 13.9% and 5.1%, respectively. In the post-vaccine period, a shift in the genetic structure of serotype 19A was found, with a significant increase of PMEN-14 (CC320) and a concurrent decrease of the major clone Denmark 14 -32 (CC230) observed prior to PCV10 introduction in Bulgaria., Conclusions: Clonality was found behind the wide distribution of MDR capsular types 19A, 6C, 23A and 3 following vaccine introduction, and a highly multiresistant and virulent clone Taiwan 19F -14/ST320 has emerged as a common pathogen in children., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

The 10-valent pneumococcal conjugate vaccine (PCV10) has been included in Bulgarian Childhood Immunization Program since 2010. This study aimed to assess serotype distribution and antimicrobial resistance of 198 invasive and non-invasive Streptococcus pneumoniae strains that had been isolated in Bulgaria during 2011-2016 from patients with invasive (IPD) and non-invasive (NIPD) pneumococcal diseases. The most common invasive serotypes were 3 (10.1%), 19F (4.0%), and 7F (3.0%). A significant decrease in the proportion of invasive vaccine types (VTs) from 64.2% to 35.2% was found in comparison with pre-vaccine era. The most common serotypes among middle ear fluids were 3, 19A and 19F (5.6% each), and VTs fell down from 66.4% to 40.0% in post-PCV10 period. Among respiratory isolates, the most prevalent serotypes were some emergent serotypes such as 15A/B/C (5.0%), 19A, and 6C (4.0% each). VTs decreased significantly (16.3%) among vaccinated children compared to unvaccinated children and adults (44.0%). Two non-VTs (19A and 6C) have increased significantly more (p<0.05) in vaccinated children than in unvaccinated patients. The rates of antibiotic nonsusceptible S. pneumoniae in Bulgaria remained high in post-PCV10 era. Among all source of isolates, antimicrobial nonsusceptibility rates were: oral penicillin - 46.5%, trimethoprim-sulfamethoxazole - 45.4%, erythromycin - 43.9%, tetracycline - 37.4%, and multidrug-resistance (MDR) was 44%. The most common MDR serotypes were 19F, 19A, 6A/C, 15A/B/C and 23A. Our results proved that PCV10 vaccination substantially reduced VTs pneumococcal IPD and NIPD. There has been a shift in the distribution of S. pneumoniae serotypes mostly in vaccinated children but also in the whole population and strong serotype-specific antibiotic resistance was observed after vaccine implementation. Therefore, it is important to continue monitoring serotype changes and pneumococcal resistance among all patient ages in addition to aid in determining the long-term effectiveness of PCV10 interventions., (Copyright © 2017 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.)

The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced as a mandatory vaccine in Bulgaria in April 2010. We report on the serotype distribution and the antimicrobial resistance of 222 invasive Streptococcus pneumoniae isolates collected from all age groups before the introduction of PCV10. PCV7, PCV10, and PCV13 covered 43.7, 59.9, and 78.8% of all invasive pneumococcal strains, and 64.2, 79.1, and 89.6% of isolates involving children less than 5 years of age. Penicillin resistance was found in 30.1% of the isolates responsible for meningitis and in 5.0% of isolates responsible for other invasive infections. Overall, erythromycin resistance was found in 19.4% of all invasive strains. The erm(B) was the most prevalent pneumococcal macrolide resistance genotype (63.2%) and dual mechanisms of both genes the erm(B) and mef(E) were detected in 15.8% of 19 erythromycin resistant isolates during the period 2006-2010. The prevalence and spread of serotypes 19F, 6B, and 19A during the last period may have contributed to the high predominance of erm(B) genotype in comparison of mef genotype, which was predominant in our country among erythromycin-resistant isolates before 2005. Continuing surveillance is required after the recent introduction of PCV10 in order to observe future developments of any serotype changes in the Bulgarian population, as well as surveillance of antimicrobial susceptibility of invasive S. pneumoniae isolates.

The aim of the study was to describe the emergence, the spread, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria. Over eight years (1996-2003), 442 ESBL-screen-positive isolates were collected in nine medical institutions in four Bulgarian towns. Class A ESBLs of the SHV, TEM, and CTX-M groups were identified in seven species. SHV-type enzymes persisted during the whole study period, TEM-ESBLs appeared first in 1999, and CTX-M-types appeared first in 2001. The rate of CTX-M enzyme producers increased rapidly between 2001 and 2003, while the rate of SHV producers decreased. Six different ESBL-types were identified, namely, SHV-2, -5, and -12, CTX-M-3 and -15, and a new TEM-3-like variant (TEM-139). The most widespread enzymes were SHV-12, CTX-M-15, and CTX-M-3 found in seven centers. TEM-139 was identified mainly in one center. A trend for strains harboring more than one ESBL gene, for example, CTX-M + SHV, was observed since 2002. Plasmid fingerprinting and random amplified polymorphic DNA analysis typing revealed wide dissemination of identical plasmids among different bacterial species and hospitals, as well as clonal spread of ESBL producers. Our data contribute to clarify the dynamics in the prevalence of ESBLs in Bulgaria and demonstrate the importance of molecular procedures for their analysis.

A total of 328 clinical isolates of Streptococcus pneumoniae were analyzed to determine the rate of macrolide and penicillin resistance as well as macrolide resistance phenotypes and genotypes. Erythromycin resistance was found in 81 pneumococcal isolates (24.7%) and 10.7% of isolates were clindamycin resistant. The prevalence of penicillin G-intermediate (minimum inhibitory concentrations, MICs, 0.125 to 1 microg/ml) and penicillin-resistant (MICs, >or=2 microg/ml) S. pneumoniae isolates was 25.6% and 13.7%, respectively. The rate of ceftriaxone-intermediate and ceftriaxone-resistant strains was 2.7% and 1.2%, respectively. Among erythromycin-resistant S. pneumoniae isolates, strains harboring mef(A) genes (n=42; 51.8%) were found to be predominant over strains with erm(B) genes (n=34; 42.0%). One (1.2%) isolate carried both erm(B) and mef(A), while 4 (4.9%) isolates carried L4 protein mutations. By using the erythromycin, clindamycin and rokitamycin triple-disk test, 42 strains were assigned to the M phenotype of macrolide resistance, 31 isolates were assigned to the partially inducible (iMcLS) phenotype, 4 were assigned to the constitutive (cMLS) phenotype. Four strains with L4 gene showed a rare phenotype with the triple-disk test. Serotyping of S. pneumoniae isolates suggested that serotype (or serogroup) 14, 6 and 19 were predominant (81.5%) among erythromycin-resistant strains. Among mef(A) positive isolates serotype 14 was predominant, among erm(B) positive isolates serogroups 6 and 19 were the most prevalent.

In total, 1039 pediatric Streptococcus pyogenes isolates from Bulgaria, Croatia, the Czech Republic, Hungary, Latvia, Lithuania, Poland, Romania, Slovakia and Slovenia were studied. All strains were susceptible to penicillin G, levofloxacin, and quinupristin-dalfopristin, 91-100% to telithromycin, and 82-100% to erythromycin, azithromycin, and clarithromycin, and 90-100% to clindamycin. Macrolide resistance occurred mainly in Slovakia (25%), the Czech Republic (17.3%), and Croatia (15.8%). Overall, 9.7% of S. pyogenes isolates were erythromycin resistant due to erm(B)- or erm(A)-encoded methylases (72.3%) or to a mef(A)-encoded efflux pump (25.7%). One strain had alterations of both 23S rRNA (A2058G Escherichia coli numbering) and ribosomal protein L22 (G95D).

Objective: To test the activity of telithromycin against 1034 Streptococcus pneumoniae isolates from pediatric patients in ten centers from ten central and eastern European countries during 2000-2001, and to compare it with the activities of erythromycin A, azithromycin, clarithromycin, clindamycin, and quinupristin-dalfopristin., Methods: The minimum inhibitory concentrations (MICs) of telithromycin, erythromycin A, azithromycin, clarithromycin, clindamycin, levofloxacin, quinupristin-dalfopristin and penicillin G were tested by the agar dilution method with incubation in air, and mechanisms of resistance to macrolides and quinolones were investigated., Results: Strains were isolated from sputum, tracheal aspirates, ear, eye, blood, and cerebrospinal fluid. Among S. pneumoniae strains tested, 36% had raised penicillin G MICs (>/= 0.12 mg/L). Susceptibilities were as follows: telithromycin, quinupristin-dalfopristin and levofloxacin, >/= 99%; clindamycin, 83%; and erythromycin A, azithromycin and clarithromycin, 78%. Of 230 (22.3%) erythromycin A-resistant S. pneumoniae strains, 176 (79.6%) had erm(B), 38 (16.1%) had mef(A), and 10 (4.3%) had mutations in 23S ribosomal RNA or in ribosomal protein L4. The rates of drug-resistant S. pneumoniae are high in all centers except Kaunas, Riga, and Prague., Conclusion: Telithromycin had low MICs against all strains, irrespective of macrolide, azalide or clindamycin resistance. Ribosomal methylation was the most prevalent resistance mechanism among all resistant strains, except in Sofia, where the prevalence of the efflux mechanism was higher.

Objectives: We conducted a study to provide information on the importance of bacterial pathogens causing childhood meningitis in Bulgaria., Methods: A 5-year population-based retrospective survey for bacterial meningitis in children <5 years of age was performed at all hospitals in the six largest regions of Bulgaria., Results: There were 297 cases of meningitis reported, of which 211 (71.0%) were classified as bacterial in origin. The most common causes were Neisseria meningitidis (49 cases) and Haemophilus influenzae type b (Hib) (44 cases), accounting for 36% and 32% of etiologically confirmed cases. Thirty-one cases (70.5%) of Hib meningitis occurred in children <2 years of age, and 26 (59.1%) occurred in children 6-23 months of age. Average annual incidence rates of Hib meningitis based on the population of children <5 years of age for each region ranged from 1.3 to 9.8 per 100,000 (mean 5.9/100,000)., Conclusion: The estimated incidence rates from this study were similar to those reported from southern European and Mediterranean countries. Further studies are planned to provide information on appropriate strategies for preventing childhood meningitis in Bulgaria.

The macrolide and levofloxacin susceptibilities of 992 isolates of Streptococcus pneumoniae from clinical specimens collected in 1999 and 2000 were determined in 10 centers in Central and Eastern European countries. The prevalences of penicillin G-intermediate (MICs, 0.125 to 1 microg/ml) and penicillin-resistant (MICs, < or =2 microg/ml) Streptococcus pneumoniae isolates were 14.3 and 16.6%, respectively. The MICs at which 50% of isolates are inhibited (MIC(50)s) and the MIC(90)s of telithromycin were 0.016 and 0.06 microg/ml, respectively; those of erythromycin were 0.06 and >64 microg/ml, respectively; those of azithromycin were 0.125 and >64 microg/ml, respectively; those of clarithromycin were 0.03 and >64 microg/ml, respectively; and those of clindamycin were 0.06 and >64 microg/ml, respectively. Erythromycin resistance was found in 180 S. pneumoniae isolates (18.1%); the highest prevalence of erythromycin-resistant S. pneumoniae was observed in Hungary (35.5%). Among erythromycin-resistant S. pneumoniae isolates, strains harboring erm(B) genes (125 strains [69.4%]) were found to be predominant over strains with mef(E) genes (25 strains [13.4%]), L4 protein mutations (28 strains [15.6%]), and erm(A) genes (2 strains [1.1%]). Similar pulsed-field gel electrophoresis patterns suggested that some strains containing L4 mutations from the Slovak Republic, Bulgaria, and Latvia were clonally related. Of nine strains highly resistant to levofloxacin (MICs, >8 microg/ml) six were isolated from Zagreb, Croatia. Telithromycin at < or =0.5 microg/ml was active against 99.8% of S. pneumoniae isolates tested and may be useful for the treatment of respiratory tract infections caused by macrolide-resistant S. pneumoniae isolates.

Among 1,011 recently isolated Streptococcus pyogenes isolates from 10 Central and Eastern European centers, the MICs at which 50% of isolates are inhibited (MIC(50)s) and the MIC(90)s were as follows: for telithromycin, 0.03 and 0.06 microg/ml, respectively; for erythromycin, azithromycin, and clarithromycin, 0.06 to 0.125 and 1 to 8 microg/ml, respectively; and for clindamycin, 0.125 and 0.125 microg/ml, respectively. Erythromycin resistance occurred in 12.3% of strains. Erm(A) [subclass erm(TR)] was most commonly encountered (60.5%), followed by mef(A) (23.4%) and erm(B) (14.5%). At <0.5 microg/ml, telithromycin was active against 98.5% of the strains tested.

Objective: To assess the incidence of meningitis caused by Haemophilus influenzae type b (Hib) among children in Bulgaria and to provide evidence for an informed decision on the use of Hib vaccines in Bulgaria., Methods: From 1 July 1997 to 31 December 1999, active surveillance for meningitis was conducted in six regions. For children with suspected meningitis, a cerebrospinal fluid (CSF) specimen was sent for cytology, chemistry, latex agglutination testing, culture and sensitivity., Findings: During the 2.5-year study period, surveillance was conducted among 138 249 children aged <5 years - a sample representing 40% of all Bulgarian children in this age group. Overall, 285 children with suspected meningitis were identified. In eight children, clinical symptoms of meningitis resolved rapidly before a CSF specimen could be obtained. Of the remaining 277 children, 121 (44%) were classified as having probable bacterial meningitis on the basis of a CSF examination. An organism was identified for 88 (73%) of the 121 cases with probable bacterial meningitis. There were 21 cases of Hib, giving a mean annual incidence of 6.1 Hib meningitis cases per 100 000 children <5 years; the case-fatality rate was 10%. Nearly 60% of Hib isolates were resistant to one or more antibiotics, but they were not resistant to third-generation cephalosporins., Conclusion: On the basis of these findings, Hib conjugate vaccines have been included in the list of vaccines recommended for children by the Bulgarian Ministry of Health. The recommended initial treatment for paediatric bacterial meningitis has been changed to third-generation cephalosporins.

As part of an ongoing surveillance program of antibiotic-resistant Streptococcus pneumoniae in Sofia, Bulgaria, 120 penicillin-resistant strains (PRSP) (most of them recovered from children hospitalized with pneumococcal disease) were analyzed by microbiological and molecular methods. Several unique features of this collection are of particular interest. (i) Most isolates (112 of 120) were also resistant to trimethoprim-sulfamethoxazole (SXT) (97 of 120 isolates, or 80%), and over 70% (86 of 120) of the isolates were resistant to at least three antibiotics in addition to penicillin. (ii) Close to 80% of all isolates were represented by large clusters of bacteria, each with a unique serotype, antibiotype, and chromosomal macrorestriction pattern (determined by pulsed-field gel electrophoresis), as well as unique restriction fragmentation length polymorphisms of the penicillin-binding protein genes pbp1a, pbp2x, and pbp2b. (iii) A large proportion (45 of 120, or 38%) of the strains belonged to two internationally spread epidemic clones of S. pneumoniae, the first expressing capsular type 23F and the second expressing serotype 9. (iv) A unique Bulgarian cluster composed of eight serotype 19F isolates was resistant to tetracycline, SXT, cefotaxime, and extremely high levels of penicillin and erythromycin. Nevertheless, this clone did not react with either the erm or the mef DNA probes, and thus the mechanism of macrolide resistance in this group of PRSP remains to be elucidated.

With use of standardized techniques, a study of nasopharyngeal pneumococcal carriage in children in six Central and Eastern European cities was undertaken during the winter of 1993-1994. Nasopharyngeal swab specimens were collected from 954 children (predominantly under the age of 5 years) who were hospitalized or attending outpatient clinics or day-care centers. Susceptibility of isolates was determined by disk diffusion (on Mueller-Hinton agar with 5% sheep blood). Disks containing 1 micrograms of oxacillin were used to screen for susceptibility to penicillin G. Pneumococci were recovered from 258 (27.0%) of the 954 children. A variety of strains were recovered, and most penicillin-resistant strains were ŕesistant to multiple agents. Minimum inhibitory concentrations of penicillin for selected resistant strains were 0.125-8 micrograms/mL. Resistance to penicillin was common in strains from Bulgaria, Romania, and Slovakia. Resistance to erythromycin and chloramphenicol occurred in Bulgarian and Romanian strains. Strains from Poland were all susceptible to penicillin, but many were resistant to tetracycline. Resistance to trimethoprim-sulfamethoxazole was common in Bulgarian, Romanian, and Slovak strains. Czech and Russian strains were predominantly susceptible to antibiotics. Most resistant strains were of serotypes 6, 14, 19, and 23.

The antimicrobial resistance of Streptococcus pneumoniae was surveyed in 1991-1993 at the Hospital for Infectious Diseases in Sofia, Bulgaria. Pneumococcal isolates were collected from routine clinical specimens and from nasopharyngeal secretions of inpatient carriers. The incidence of penicillin-resistant S. pneumoniae (PRSP) was 24.3% among clinical samples and nasopharyngeal carriage of PRSP was as high as 40% among children. Penicillin-resistant strains were more frequently resistant to non-beta-lactam antibiotics than were penicillin-sensitive strains. More than half of the PRSP strains were multiply resistant. On the basis of MIC values of ampicillin, it was established that ampicillin was not superior to penicillin. The MICs of five cephalosporins were found to increase in parallel with the MICs of penicillin G. Some of the pneumococcal strains that were highly penicillin-resistant were also resistant to cefotaxime/ceftriaxone (MIC = 1-4 micrograms/ml), but the number of strains was small. On the basis of MIC values of cefotaxime and ceftriaxone for strains from cerebrospinal fluid, both antibiotics may be suitable alternatives for treating meningitis due to strains with resistance to penicillin.