Phenotypic and genotypic characteristics of non-invasive S. pneumoniae isolates recovered from PCV10-vaccinated children in Bulgaria.

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 â€‹= â€‹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.

Rise of multidrug-resistant Streptococcus pneumoniae clones expressing non-vaccine serotypes among children following introduction of the 10-valent pneumococcal conjugate vaccine in Bulgaria.

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 (Taiwan19F-14), CC315 (Poland6B-20) and CC180 (Netherlands3-31), 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 Denmark14-32 (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 Taiwan19F-14/ST320 has emerged as a common pathogen in children.

Serotype distribution and antimicrobial resistance of invasive Streptococcus pneumoniae isolates in Bulgaria before the introduction of pneumococcal conjugate vaccine.

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.

Prevalence and macrolide resistance phenotypes and genotypes among clinical isolates of Streptococcus pneumoniae collected in Sofia, Bulgaria from 2001 to 2005.

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.

Activity of telithromycin and seven other agents against 1034 pediatric Streptococcus pneumoniae isolates from ten central and eastern European centers.

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.

Activity of telithromycin compared with seven other agents against 1039 Streptococcus pyogenes pediatric isolates from ten centers in central and eastern Europe.

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).

Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates from Bulgaria.

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.

Carriage of antibiotic-resistant Streptococcus pneumoniae by children in eastern and central Europe--a multicenter study with use of standardized methods.

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 resistant 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.

Clinical isolates and nasopharyngeal carriage of antibiotic-resistant Streptococcus pneumoniae in Hospital for Infectious Diseases, Sofia, Bulgaria, 1991-1993.

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.