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Background: Widespread use of pneumococcal conjugate vaccines (PCVs) has reduced vaccine-type invasive pneumococcal disease (IPD). We describe the serotype distribution of IPD after extensive use of ten-valent PCV (PCV10; Synflorix, GSK) and 13-valent PCV (PCV13; Prevenar 13, Pfizer) globally., Methods: IPD data were obtained from surveillance sites participating in the WHO-commissioned Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project that exclusively used PCV10 or PCV13 (hereafter PCV10 and PCV13 sites, respectively) in their national immunisation programmes and had primary series uptake of at least 70%. Serotype distribution was estimated for IPD cases occurring 5 years or more after PCV10 or PCV13 introduction (ie, the mature period when the serotype distribution had stabilised) using multinomial Dirichlet regression, stratified by PCV product and age group (<5 years, 5-17 years, 18-49 years, and ≥50 years)., Findings: The analysis included cases occurring primarily between 2015 and 2018 from 42 PCV13 sites (63 362 cases) and 12 PCV10 sites (6806 cases) in 41 countries. Sites were mostly high income (36 [67%] of 54) and used three-dose or four-dose booster schedules (44 [81%]). At PCV10 sites, PCV10 serotypes caused 10·0% (95% CI 6·3-12·9) of IPD cases in children younger than 5 years and 15·5% (13·4-19·3) of cases in adults aged 50 years or older, while PCV13 serotypes caused 52·1% (49·2-65·4) and 45·6% (40·0-50·0), respectively. At PCV13 sites, PCV13 serotypes caused 26·4% (21·3-30·0) of IPD cases in children younger than 5 years and 29·5% (27·5-33·0) of cases in adults aged 50 years or older. The leading serotype at PCV10 sites was 19A in children younger than 5 years (30·6% [95% CI 18·2-43·1]) and adults aged 50 years or older (14·8% [11·9-17·8]). Serotype 3 was a top-ranked serotype, causing about 9% of cases in children younger than 5 years and 14% in adults aged 50 years or older at both PCV10 and PCV13 sites. Across all age and PCV10 or PCV13 strata, the proportion of IPD targeted by higher-valency PCVs beyond PCV13 was 4·1-9·7% for PCV15, 13·5-36·0% for PCV20, 29·9-53·8% for PCV21, 15·6-42·0% for PCV24, and 31·5-50·1% for PCV25. All top-ten ranked non-PCV13 serotypes are included in at least one higher-valency PCV., Interpretation: The proportion of IPD due to serotypes included in PCVs in use was low in mature PCV10 and PCV13 settings. Serotype distribution differed between PCV10 and PCV13 sites and age groups. Higher-valency PCVs target most remaining IPD and are expected to extend impact., Funding: Bill & Melinda Gates Foundation as part of the WHO Pneumococcal Vaccines Technical Coordination Project., Competing Interests: Declaration of interests MDK reports grants from Merck and Pfizer and personal fees from Merck. KH reports employment at Pfizer from Oct 26, 2020. AvG reports funding from Pfizer and attendance at advisory board meetings for Pfizer and Merck. MvdL reports support, membership of advisory boards, and speakers honoraria from Pfizer and Merck. NMvS reports speaker and service fees from MSD and GSK, and holding a patent (WO 2013/020090 A3) with royalties paid to herself and to the University of California San Diego (inventors: Nina van Sorge and Victor Nizet). IY reports membership of an mRNA-1273 study group, and funding to her institution from BioFire, MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Novavax, Sanofi Pasteur, and Micron. EV reports grants from the French Public Health Agency, Pfizer, and Merck. JAS reports grants from the Bill & Melinda Gates Foundation, the Wellcome Trust, the UK Medical Research Council, and the National Institute for Health and Care Research. CM-A reports grants from Pfizer and speaker fees from Pfizer and MSD. AM reports research support to her institution from Pfizer and Merck, and honoraria for advisory board membership from GSK, Merck, and Pfizer. SNL reports contract research for GSK, Pfizer, and Sanofi Pasteur on behalf of St George's University of London, with no personal remuneration. NPK reports research support from Pfizer, GSK, Sanofi Pasteur, Merck, and Protein Sciences (now Sanofi Pasteur). JDK reports an unrestricted grant-in-aid from Pfizer Canada. MH reports reimbursement for advisory boards from MSD; and an investigator-initiated research grant paid to his institution from Pfizer. LLH reports research grants to her institution from GSK, Pfizer, and Merck. SD reports a grant from Pfizer. RD reports grants and research support from Pfizer, MSD, and MedImmune; consultancy for Pfizer, MeMed, MSD, BiondVax, and GSK; participation on advisory boards for Pfizer, MSD, BiondVax, and GXRD; and having been a speaker for Pfizer, AstraZeneca, and GSK. MC reports a professional fee, an unrestricted research grant, and an Investigator Initiated Reward (W1243730) from Pfizer Ireland. KA reports a grant from Merck. SCGA reports a travel grant from Pfizer. KGK reports grants from GSK. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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%). β-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.

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.

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.

Serotype-specific surveillance for invasive pneumococcal disease (IPD) is essential for assessing the impact of 10- and 13-valent pneumococcal conjugate vaccines (PCV10/13). The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project aimed to evaluate the global evidence to estimate the impact of PCV10/13 by age, product, schedule, and syndrome. Here we systematically characterize and summarize the global landscape of routine serotype-specific IPD surveillance in PCV10/13-using countries and describe the subset that are included in PSERENADE. Of 138 countries using PCV10/13 as of 2018, we identified 109 with IPD surveillance systems, 76 of which met PSERENADE data collection eligibility criteria. PSERENADE received data from most (n = 63, 82.9%), yielding 240,639 post-PCV10/13 introduction IPD cases. Pediatric and adult surveillance was represented from all geographic regions but was limited from lower income and high-burden countries. In PSERENADE, 18 sites evaluated PCV10, 42 PCV13, and 17 both; 17 sites used a 3 + 0 schedule, 38 used 2 + 1, 13 used 3 + 1, and 9 used mixed schedules. With such a sizeable and generally representative dataset, PSERENADE will be able to conduct robust analyses to estimate PCV impact and inform policy at national and global levels regarding adult immunization, schedule, and product choice, including for higher valency PCVs on the horizon.

Pneumococcal conjugate vaccine (PCV) introduction has reduced pneumococcal meningitis incidence. The Pneumococcal Serotype Replacement and Distribution Estimation (PSERENADE) project described the serotype distribution of remaining pneumococcal meningitis in countries using PCV10/13 for least 5-7 years with primary series uptake above 70%. The distribution was estimated using a multinomial Dirichlet regression model, stratified by PCV product and age. In PCV10-using sites ( N = 8; cases = 1141), PCV10 types caused 5% of cases <5 years of age and 15% among ≥5 years; the top serotypes were 19A, 6C, and 3, together causing 42% of cases <5 years and 37% ≥5 years. In PCV13-using sites ( N = 32; cases = 4503), PCV13 types caused 14% in <5 and 26% in ≥5 years; 4% and 13%, respectively, were serotype 3. Among the top serotypes are five (15BC, 8, 12F, 10A, and 22F) included in higher-valency PCVs under evaluation. Other top serotypes (24F, 23B, and 23A) are not in any known investigational product. In countries with mature vaccination programs, the proportion of pneumococcal meningitis caused by vaccine-in-use serotypes is lower (≤26% across all ages) than pre-PCV (≥70% in children). Higher-valency PCVs under evaluation target over half of remaining pneumococcal meningitis cases, but questions remain regarding generalizability to the African meningitis belt where additional data are needed.

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.

A pneumococcal conjugate vaccine (PCV10) was introduced in Bulgarian national immunization program since April 2010. Clonal composition based on pulsed-field gel electrophoresis and multilocus sequence typing genotyping of 52 serotype 19A Streptococcus pneumoniae isolates was analyzed. These were invasive and respiratory isolates collected between 1992 and 2013 from both children (78.8% <5 years) and adults with pneumococcal infections. Multidrug resistance was found in 82.7% of all 19A isolates. The most prevalent genotype (63.5%) among serotype 19A pneumococcal strains was the multidrug-resistant clonal complex CC230, which is a capsular switched variant of the Denmark(14)-32 (ST230) global clone. The most frequent sequence type (ST) was ST230 (48.1%) and together with four other closely related STs (15.4%), belonging to ST1611, ST276, ST7466, and ST2013, which were single- and double-locus variants; they were included in the main CC230. The disappearance of highly drug-resistant ST663 clone and emergence of new clones as CC320 and CC199 was also observed among the rest 19A isolates. A comparison of clonal composition between invasive and noninvasive isolates did not show a great genetic diversity among both kinds of isolates. Continuous surveillance of serotype 19A population following the introduction of PCV10 is essential to evaluate the impact of the vaccine on the epidemiology of this serotype.

Background: Pneumococcal and Haemophilus influenzae type b (Hib) vaccines were introduced in our national immunisation program in April 2010. The aims of this retrospective, laboratory-based study were to determine the serotypes and antibiotic resistance of Streptococcus pneumoniae and H. influenzae isolates from middle ear fluid (MEF) collected before the introduction of immunization., Methods: S. pneumoniae (n = 128) and H. influenzae (n = 40) strains isolated from MEF of children with AOM between 1994 and 2011 were studied. MICs were determined by a microdilution assay. Serotyping of S. pneumoniae was done by Quellung method and PCR capsular typing was used for H. influenzae. Macrolide resistance genes were detected by PCR for erythromycin resistant S. pneumoniae (ERSP). DNA sequencing of ftsI gene was performed for ampicillin nonsusceptible H. influenzae., Results: The most common serotypes found among children with pneumococcal AOM were 19 F (20.3%), 6B (15.6%), and 19A (10.9%). The potential coverage rates by the PCV7, PCV10 and PCV13 of children aged < 5 years were 63.6%, 66.4% and 85.5%, respectively. Reduced susceptibility to oral penicillin was seen in 68.1%; resistance to erythromycin was 46.9%. We found erm(B) gene in 56.7% of the ERSP, mef(E) gene in 25%; 15% harbored both genes erm(B) + mef(E) and 3.3% had mutations of L4 ribosomal protein. Of the 40 H. influenzae isolates 97.5% were nontypeable. Nonsusceptibility to ampicillin occurred in 25%. Ampicillin resistance groups were: β-lactamase-positive ampicillin resistant (BLPAR) strains (10%), β-lactamase-negative ampicillin resistant (BLNAR) strains (12.5%) and β-lactamase-positive amoxicillin-clavulanate resistant (BLPACR) strains (2.5%). Among BLNAR and BLPACR most of the isolates (5/6) belonged to group II, defined by the Asn526Lys substitution., Conclusions: The levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe AOM in children are high in our settings. The existence of multidrug-resistant S. pneumoniae serotype 19A is of particular concern. The rate of BLNAR and BLPACR strains among H. influenzae isolates was 15%.

Objective: To determine the serotypes, antimicrobial susceptibility, and beta-lactam resistance mechanisms of Haemophilus influenzae strains isolated from invasive and respiratory tract infections (RTIs) prior to the introduction of Haemophilus influenzae type b (Hib) vaccination in Bulgaria., Methods: A total of 259 isolates were serotyped by polymerase chain reaction. Susceptibility to antibiotics and beta-lactamase production were determined, and DNA sequencing of the ftsI gene was performed for ampicillin non-susceptible strains., Results: The invasive H. influenzae infections in children were mainly due to serotype b (94.5% in meningitis and 88.9% in other invasive cases). Non-typeable strains (97.4%) were the most frequently found H. influenzae strains in RTIs both in children and adults. Non-susceptibility to ampicillin occurred in 22% of all strains. Ceftriaxone and levofloxacin were the most active agents tested. Ampicillin resistance occurred in 34.4% of invasive strains, and beta-lactamase production was the only mechanism found. Among respiratory tract isolates, ampicillin non-susceptible strains (18%) were classified into the following groups: beta-lactamase-positive, ampicillin-resistant (BLPAR) strains (7.2%); beta-lactamase-negative, ampicillin-non-susceptible (BLNAR) strains (8.2%); and beta- lactamase-positive, amoxicillin-clavulanate-resistant (BLPACR) strains (2.6%). Among 21 BLNAR and BLPACR strains there were 9 different patterns of multiple-amino acid substitutions in penicillin-binding protein 3. Of these, most isolates (81.0%) belonged to group II, defined by the Asn526Lys substitution., Conclusions: Beta-lactamase production was more common among invasive strains than in respiratory isolates. BLNAR and BLPACR H. influenzae were found only among respiratory tract isolates.

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.

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.