Effect of Vaccination on Pneumococci Isolated from the Nasopharynx of Healthy Children and the Middle Ear of Children with Otitis Media in Iceland Sigríður J. Quirk,a,b,c Gunnsteinn Haraldsson,a,b,c Helga Erlendsdóttir,a,b,c Martha Á. Hjálmarsdóttir,a,b,c Andries J. van Tonder,d Birgir Hrafnkelsson,e Samuel Sigurdsson,a Stephen D. Bentley,d Ásgeir Haraldsson,a,f Angela B. Brueggemann,g,h Karl G. Kristinssona,b,c aUniversity of Iceland, Faculty of Medicine, Reykjavík, Iceland bLandspitali University Hospital, Department of Clinical Microbiology, Reykjavík, Iceland cBioMedical Centre of the University of Iceland, Reykjavik, Iceland dParasites and Microbes, Wellcome Sanger Institute, Hinxton, United Kingdom eUniversity of Iceland, Department of Mathematics, Reykjavik, Iceland fChildren's Hospital Iceland, Reykjavík, Iceland gNuffield Department of Medicine, University of Oxford, Oxford, United Kingdom hDepartment of Medicine, Imperial College London, London, United Kingdom ABSTRACT Vaccination with pneumococcal conjugate vaccines (PCVs) disrupts the pneumococcal population. Our aim was to determine the impact of the 10-valent PCV on the serotypes, genetic lineages, and antimicrobial susceptibility of pneumo- cocci isolated from children in Iceland. Pneumococci were collected between 2009 and 2017 from the nasopharynges of healthy children attending 15 day care centers and from the middle ears (MEs) of children with acute otitis media from the greater Reykjavik capital area. Isolates were serotyped and tested for antimicrobial suscepti- bility. Whole-genome sequencing (WGS) was performed on alternate isolates from 2009 to 2014, and serotypes and multilocus sequence types (STs) were extracted from the WGS data. Two study periods were defined: 2009 to 2011 (PreVac) and 2012 to 2017 (PostVac). The overall nasopharyngeal carriage rate was similar be- tween the two periods (67.3% PreVac and 61.5% PostVac, P � 0.090). Vaccine-type (VT) pneumococci decreased and nonvaccine-type (NVT) pneumococci (serotypes 6C, 15A, 15B/C, 21, 22F, 23A, 23B, 35F, and 35B) significantly increased in different age strata post-PCV introduction. The total number of pneumococci recovered from ME samples significantly decreased as did the proportion that were VTs, although NVT pneumococci (6C, 15B/C, 23A, and 23B) increased significantly. Most serotype 6C pneumococci were multidrug resistant (MDR). Serotype 19F was the predominant se- rotype associated with MEs, and it significantly decreased post-PCV introduction: these isolates were predominantly MDR and of the Taiwan19F-14 PMEN lineage. Overall, the nasopharyngeal carriage rate remained constant and the number of ME-associated pneumococci decreased significantly post-PCV introduction; however, there was a concomitant and statistically significant shift from VTs to NVTs in both collections of pneumococci. KEYWORDS Iceland, Streptococcus pneumoniae, carriage, epidemiology, molecular epidemiology, otitis media, pneumococcus, vaccination, vaccine Streptococcus pneumoniae is an important human pathogen that can cause relatively mild upper respiratory tract infections, such as acute otitis media (AOM), or more severe infections, such as pneumonia and invasive pneumococcal disease (IPD) (1). Pneumococci frequently colonize the nasopharynges of humans, especially children Received 27 June 2018 Returned for modification 24 July 2018 Accepted 20 September 2018 Accepted manuscript posted online 26 September 2018 Citation Quirk SJ, Haraldsson G, Erlendsdóttir H, Hjálmarsdóttir MÁ, van Tonder AJ, Hrafnkelsson B, Sigurdsson S, Bentley SD, Haraldsson Á, Brueggemann AB, Kristinsson KG. 2018. Effect of vaccination on pneumococci isolated from the nasopharynx of healthy children and the middle ear of children with otitis media in Iceland. J Clin Microbiol 56:e01046-18. https://doi.org/10.1128/JCM .01046-18. Editor Daniel J. Diekema, University of Iowa College of Medicine Copyright © 2018 Quirk et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Karl G. Kristinsson, Karl@landspitali.is. EPIDEMIOLOGY crossm December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 1Journal of Clinical Microbiology D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://doi.org/10.1128/JCM.01046-18 https://doi.org/10.1128/JCM.01046-18 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ mailto:Karl@landspitali.is https://crossmark.crossref.org/dialog/?doi=10.1128/JCM.01046-18&domain=pdf&date_stamp=2018-9-26 https://jcm.asm.org attending day care centers (DCCs) (2, 3), and asymptomatic carriage occurs at least once prior to the age of 2 years (4). Nasopharyngeal colonization precedes pneumococcal disease, is a major factor for horizontal transmission within the community, especially among young children (5), and reflects the pneumococcal strains circulating in the community (6). Pneumococci are among the most frequent causes of bacterial AOM (7–9), which is the most common bacterial infection in children under 3 years of age (10). Pneumococcal conjugate vaccines (PCVs) interrupt the transmission of antibiotic- resistant pneumococci and thus decrease the burden of disease caused by antibiotic- resistant isolates in immunized children. Therefore, studies of the impact of these vaccines on antibiotic resistance and serotype distribution should focus both on pneumococci from nasopharyngeal carriage and from middle ears of children of similar ages with AOM (11). Current PCVs target only a limited number of serotypes, especially those commonly causing IPD in young children (12–14). PCVs have been implemented in the infant vaccine immunization program in over 100 countries (15), which has resulted in a decrease of IPD caused by vaccine serotypes (VTs) in vaccinated children and other age groups due to herd immunity (16–18). Among healthy children, PCV implementation has also resulted in serotype replacement in carriage where VTs have been replaced with nonvaccine serotypes, meaning that the total carriage rate has remained constant (19–21). Pneumococcal carriage has been monitored and recorded in Iceland for the past 2 decades (22–24). The carriage rates have been consistent over time, ranging from 50% to 70% in healthy preschool children 1 to �7 years of age (25). The 10-valent PCV (PHiD-CV [Synflorix; GSK]), which directly targets 10 serotypes, was introduced into the national pediatric immunization program in Iceland in April 2011 in a 2-plus-1 schedule without catch-up. No other pneumococcal vaccine was previously included. The aim of this study was to assess the impact of PHiD-CV on the distribution of pneumococcal serotypes and genetic lineages among pneumococci from the nasopharynges of healthy children and the middle ears of children with otitis media and assess any changes in antimicrobial resistance rates after vaccine implementation. MATERIALS AND METHODS DCC study and bacterial isolates. Nasopharyngeal swabs were taken from healthy children 1 to �7 years of age in March every year from 2009 to 2017 after informed consent was obtained from the parents. The children were attending 15 DCCs, chosen to be representative of the greater Reykjavík area. The period 2009 to 2011 was defined as the prevaccination period (PreVac), and the years 2012 to 2017 were the postvaccine implementation period (PostVac). The number of nasopharyngeal swabs taken from each age group is listed in Table S1 in the supplemental material. The samples were selectively cultured for pneumococci on blood agar containing 5 �g/ml gentamicin and incubated anaerobically (26). Very few children attended public DCCs before the age of 12 months, and no children �1 year of age were sampled in this study. Middle ear study and bacterial isolates. All pneumococci isolated from middle ear (ME) samples from children 0 to �7 years of age with otitis media submitted to the Department of Clinical Microbi- ology, Landspitali University Hospital, between 1 January 2009 and 30 September 2017 were included in this study. This is the primary microbiology laboratory for the greater Reykjavík capital area. It provides services for individuals from the rural area and visiting specialists in the capital, and it is the reference laboratory for the whole country (approximately 85% of the population for ME samples). The primary service area for the Landspitali University Hospital was considered to be within 100 km driving distance from the hospital, and the population demographic information for this referral region was obtained from Statistics Iceland (www.statice.is). The average population sizes for children 0 to �7 years of age in the referral region during the study period were 23,747 children PreVac and 24,083 children PostVac (approximately 66% of all children in Iceland 0 to �7 years of age). A detailed listing of the populations according to age groups can be seen in Table S2. When two or more pneumococcal isolates of the same phenotype were identified from the same patient within 30 days, only one isolate was included in the analyses. Serotyping. Serotypes were determined for all available isolates with the Immulex pool antisera (State Serum Institute, Copenhagen, Denmark) and/or by multiplex PCR (mPCR), according to previously published methods (27–30). The mPCR scheme included 78 sets of serogroup/serotype-specific primer pairs and two primer pairs for a positive internal control: cpsA for the capsular locus and lytA for autolysin. Serotypes of serogroup 6 were identified using previously described PCR methods (31–33). Nontypeable (nonencapsulated S. pneumoniae [NESp]) isolates, i.e., those that were negative for cpsA and positive for lytA, were tested for the cpsB gene, which is essential for capsulation (34), according to a previously published PCR method (35). For those pneumococcal genomes that were sequenced, seqSerotyper Quirk et al. Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 2 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. http://www.statice.is https://jcm.asm.org (https://github.com/avantonder/seqSerotyper) was used to extract the serotypes from sequence data (36). DNA extraction and whole-genome sequencing. Every other pneumococcal isolate from naso- pharyngeal and ME samples from the period 2009 to 2014 was selected for whole-genome sequencing (WGS). DNA was extracted using the Promega Maxwell 16 platform, and the DNA extracts were sequenced on the Illumina HiSeq 2000. WGS data were assembled using Velvet (37) before SSPACE and GapFiller were used to improve the assemblies and close gaps (38, 39). The final assembled genomes were uploaded into a Bacterial Isolate Genome Sequence Database (BIGSdb) along with associated metadata (40). The multilocus sequence type (ST) of each isolate was extracted from the WGS data using BIGSdb and the PubMLST database (http://pubmlst.org/spneumoniae/). STs were assigned to clonal complexes (CCs) via Phyloviz (41). Genomic analyses. Prokka was used to predict the coding sequences in each genome (42). The resulting annotation files in gff format were then used as the input for Roary and clustered using a sequence identity threshold of 90% (43). The core genome was estimated using a Bayesian core genome model and a threshold of 99.9% for nasopharyngeal isolates and 99.8% for ME isolates (44). The core genes in both sample groups were extracted and aligned using MAFFT (45). FastTree was used to construct phylogenetic trees, and ClonalFrameML (46) was used to reconstruct the trees to account for recombination. The final phylogenetic trees were then annotated using iTOL (47). Antibiotic susceptibility testing. All isolates were tested for antimicrobial susceptibility to chlor- amphenicol, erythromycin, tetracycline, trimethoprim-sulfamethoxazole, and clindamycin by disk diffu- sion tests. Oxacillin disks (1 �g) were used to screen for penicillin resistance; isolates susceptible to oxacillin were considered to be susceptible to penicillin. For oxacillin-resistant isolates, the MICs to penicillin and ceftriaxone (ME isolates only) were measured using the Etest (bioMérieux, France) (48). Penicillin-nonsusceptible pneumococci (PNSP) were defined as isolates having a MIC of �0.06 mg/liter, while resistant pneumococci were isolates having a MIC of �2 mg/liter. Multidrug resistance (MDR) was defined as nonsusceptibility to three or more classes of antimicrobials (regardless of penicillin nonsus- ceptibility). The susceptibility testing was performed according to the methods and criteria of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (49). Statistical analyses. A likelihood ratio test (50) was used to test the null hypothesis of equal rates when comparing the rate (r1) of a certain serotype, CC, or ST in a given age group PreVac to the rate (r2) of the same serotype, CC, or ST in the same age group PostVac. The test is based on the assumption that the counts of the serotype, CC, or ST in the two periods are independent and both follow Poisson distributions. It is assumed that the mean of the counts PreVac is equal to the rate r1 times the total number of individuals in the age group PreVac. The mean of the counts PostVac is equal to the rate r2 times the total number of individuals in the age group PostVac. The asymptotic distribution of the likelihood ratio test statistics is a chi-square distribution with one degree of freedom under the null hypothesis. Microsoft Excel was used to calculate the test statistics and the corresponding P values. The two-sided Fisher’s exact test was used to calculate the P values for antimicrobial resistance by using the statistical software R, version 3.3.2. The level of significance for all tests was �0.05. The Simpson diversity index was used to calculate the diversity of STs (51). Ethics. The study was approved by The National Bioethics Committee (VSNb2013010015/03.07) and the appropriate authorities at the Landspitali University Hospital and the day care centers. RESULTS Nasopharyngeal samples from children attending DCCs. A total of 4,461 naso- pharyngeal swabs were collected (450 to 550 samples each year): 1,380 PreVac and 3,081 PostVac (Table 1). The median age of the children who were sampled was 4.1 years. The cultures yielded 3,029 pneumococcal isolates, and 250 children carried two pneumococcal strains. Nine isolates were excluded (not viable/not stored), yielding 3,020 isolates: 991 (32.8%) PreVac and 2,029 (67.2%) PostVac. The carriage rates were 67.3% PreVac and 61.5% PostVac (P � 0.090). Overall, 51.8% (n � 1,563) of the isolates were collected from children 4 to �7 years of age, and the fewest isolates, 3.5% (n � 103), were from the children 1 to �2 years of age (see Table S3 in the supplemental material). The genomes of 987 (49.2%) pneumococcal isolates from 2009 to 2014 were sequenced. Serotypes. A total of 36 different serotypes were detected: 27 PreVac and 35 PostVac. Overall, the numbers of isolates of serotypes included in PHiD-CV (vaccine type [VT]) decreased between the two periods (P � 0.001) (Table 1). VT pneumococci were most common in 2010 (171/324 [52.8%]) and least common in 2016 (5/354 [1.4%]) (Table 1). The numbers of isolates of serotypes not included in PHiD-CV (nonvaccine type [NVT]) increased from PreVac (n � 545, 395.2/1,000 samples) to PostVac (n � 1,725, 559.9/1,000 samples; P � 0.001) (Table 1). NVT pneumococci were most common in 2016 (349/354 [98.6%]) and least common in 2010 (153/324 [47.2%]) (Table 1). In children 1 to �2 years of age, only one NVT, serotype 23A, increased from PreVac (n � 0, 0/1,000 samples) to PostVac (n � 7, 95.9/1,000 samples; P � 0.005). The NVTs Vaccination: Pneumococci from Carriage and Middle Ear Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 3 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://github.com/avantonder/seqSerotyper http://pubmlst.org/spneumoniae/ https://jcm.asm.org that increased PostVac and were the most prevalent in children 2 to �4 years of age were of serotypes 6C (n � 82, 65.0/1,000 samples; P � 0.001), 15B/C (n � 81, 64.2/1,000 samples; P � 0.001), and 23B (n � 69, 54.7/1,000 samples; P � 0.001) (Table S3). The NVTs that increased PostVac in children 4 to �7 years of age were serotypes 23B (n � 80, 45.8/1,000 samples; P � 0.001), 6C (n � 56, 32.1/1,000 samples; P � 0.001), and 21 (n � 53, 30.4/1,000 samples; P � 0.001). Serotype 23B was only detected PostVac among children 4 to �7 years of age (Table S3). MLST/CC. Among the 987 sequenced isolates, 47 CCs (35 CCs PreVac and 41 CCs PostVac) and 104 STs (66 STs PreVac and 83 STs PostVac) were detected, and 12 CCs and 43 STs were unique to nasopharyngeal isolates. The Simpson diversity index of the STs was 0.97 for both periods. A phylogenetic tree was created with the concatenated sequences of 1,066 full-length coding loci found in 99.9% of the nasopharyngeal TABLE 1 Serotype distribution each study year in nasopharyngeal samples among children 1 to �7 years old PreVac (2009 to 2011) and PostVac (2012 to 2017) Serotype or sample type No. of isolates 2009 to 2011 (n/1,000 samples) No. of isolates PostVac 2012 to 2017 (n/1,000 samples) P value2009 2010 2011 2012 2013 2014 2015 2016 2017 PreVac 3 18 26 24 11 13 20 32 25 12 68 49.3 113 36.7 0.052 4 2 0 0 0 0 0 0 0 0 2 1.4 0 0 0.096 6A 38 24 25 56 23 16 21 23 11 87 63.0 150 48.7 0.051 6B 50 45 14 17 16 26 9 1 2 109 79.0 71 23.0 �0.001 6C 3 4 2 0 11 7 31 49 53 9 6.5 151 49.0 �0.001 9V 12 3 3 1 0 0 0 0 0 18 13.0 1 0.3 �0.001 9A 0 0 1 0 1 0 0 0 0 1 0.7 1 0.3 0.619 9N 6 0 3 3 4 6 5 1 0 9 6.5 19 6.2 0.873 10 0 0 0 0 0 0 3 5 0 0 0 8 2.6 0.052 10A 5 0 0 2 1 8 3 1 8 5 3.6 23 7.5 0.131 10B 0 0 0 0 0 7 0 0 0 0 0 7 2.3 0.075 11A 18 19 21 22 10 38 37 24 5 58 42.0 136 44.1 0.756 13 0 0 0 0 1 0 0 0 0 0 0 1 0.3 0.691 14 26 36 12 8 9 9 3 0 0 74 53.6 29 9.4 �0.001 15 0 0 1 0 0 0 1 1 1 1 0.7 4 1.3 0.385 15A 0 0 0 0 0 1 14 11 9 0 0 35 11.4 �0.001 15B/C 21 6 10 29 27 29 24 22 15 37 26.8 146 47.4 �0.001 16F 3 8 13 3 2 4 5 5 6 24 17.4 25 8.1 0.008 17 0 0 0 0 0 0 1 0 0 0 0 1 0.3 0.691 18C 0 0 0 0 0 0 2 0 0 0 0 2 0.6 0.477 19 15 12 4 7 6 3 3 0 0 31 22.5 19 6.2 �0.001 19F 30 28 29 22 28 21 13 3 0 87 63.0 87 28.2 �0.001 19A 47 16 26 12 26 29 22 31 25 89 64.5 145 47.1 0.020 21 2 1 2 4 15 17 14 23 13 5 3.6 86 27.9 �0.001 22F 3 2 2 3 17 31 4 9 5 7 5.1 69 22.4 �0.001 23 0 0 0 0 0 0 1 2 1 0 0 4 1.3 0.227 23F 50 47 28 39 30 7 17 1 3 125 90.6 97 31.5 �0.001 23A 12 10 7 6 22 24 16 35 6 29 21.0 109 35.4 0.009 23B 1 0 0 9 15 25 41 34 28 1 0.7 152 49.3 �0.001 24F 0 0 0 0 0 0 0 0 4 0 0 4 1.3 0.227 29 3 0 0 2 0 0 0 0 0 3 2.2 2 0.6 0.210 31 2 0 0 0 0 0 0 1 5 2 1.4 6 1.9 0.765 33 2 2 1 4 2 4 0 1 0 5 3.6 11 3.6 0.954 33F 3 2 5 1 1 2 4 8 1 10 7.2 17 5.5 0.492 33_Hybrid 0 0 0 0 0 4 0 0 0 0 0 4 1.3 0.227 35F 0 0 0 3 15 18 16 4 0 0 0 56 18.2 �0.001 35B 1 1 4 0 17 12 13 2 13 6 4.3 57 18.5 �0.001 38 0 9 10 2 5 3 3 1 3 19 12.8 17 5.5 0.007 Other serotypesa 0 1 1 0 1 1 11 17 19 2 1.4 49 15.9 �0.001 NESpb 21 22 25 18 16 22 22 14 24 68 49.3 116 37.7 0.075 Total 394 324 273 284 334 394 391 354 272 991 718.1 2,029 658.6 �0.001 VTc 185 171 90 94 89 66 45 5 5 446 323.2 304 98.7 �0.001 NVTd 209 153 183 190 245 328 346 349 267 545 394.9 1,725 559.9 �0.001 All NPe samples 516 444 420 465 471 566 533 540 506 1380 3,081 Samples of positive Pnf (%) 76.4 73.0 65.0 61.1 70.9 69.6 73.4 65.6 53.8 71.8 65.9 aSerotypes other than those included in the multiplex PCR panel of the study. bNESp, nonencapsulated S. pneumoniae. cSerotypes detected in the study that are included in PHiD-CV (4, 6B, 9V, 14, 18C, 19F, and 23F). dSerotypes that are not included in PHiD-CV. eNP, nasopharyngeal samples. fPn, pneumococci. Quirk et al. Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 4 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://jcm.asm.org carriage pneumococcal genomes. The tree was annotated with CC designations and serotypes (Fig. 1). CC43923F/A/B was the most common CC in both study periods. Initially, 77.6% of the isolates of CC43923F/A/B were VT serotype 23F, but PostVac, 42.0% were NVT serotype 23B; however, serotype 23B was rare prior to vaccine introduction (Fig. 1 and Table S4). Between the two periods, the prevalence of CC43322F increased from two isolates (1.5/1,000 samples) to 30 (20.0/1,000 samples; P � 0.001) (Fig. 1 and Table S4), and similarly, CC126215B/C increased from three isolates (2.2/1,000 samples) to 18 (12.0/ 1,000 samples; P � 0.032) (Fig. 1 and Table S4). Antimicrobial resistance. The overall prevalence of PNSP among nasopharyngeal isolates did not change significantly between the two study periods: 15.0% (n � 149/991) versus 16.7% (338/2,029; P � 0.268) (Table 2). Erythromycin-resistant pneu- mococci decreased from 17.6% to 13.7% (P � 0.007). The overall prevalence of MDR pneumococci decreased slightly but significantly between the periods, from 15.2% (n � 151/991) to 12.4% (n � 251/2,029; P � 0.030) (Table 2). Before PHiD-CV was introduced, 85.2% of PNSP pneumococci were also MDR, and this was reduced to 70.1% (P � 0.001). The most prevalent PNSP and MDR isolates were of serotype 19F PreVac and NESp PostVac (Table 2). Among the PNSP serotype 19F isolates, 92.5% belonged to CC236/271/32019F (Fig. 1 and Table S4). Serotype 19A PNSP increased from 4.0% to 10.4% of isolates (P � 0.021) (Table 2), and the isolates were predominantly members of CC19919A,15B/C (Fig. 1 and Table S4). PNSP with serotypes 6C (also MDR) and 35B were also detected. VICE1319a-6B V IC E 15 65 -2 2F V IC E 09 02 b- 23 A V IC E 14 57 -2 3A VICE1796-6A VICE1529-19F 3-8451 E CI V V IC E 14 75 -2 3F 3-2811 E CI V VICE1314a-6B VICE1831-33_Hybrid VICE0932-6B V IC E 1882b-N E S p VIC E12 07 -2 3A VICE0998-6B VICE1544-19A 3- 82 90 E CI V VICE1463-6C V IC E 1491b-N E S p VICE1538-6B V IC E 08 56 -6 B ii V IC E 15 47 -6 C V IC E 18 05 -2 2F VICE1852-11A V IC E 1277-19A VICE0875-23F VICE1281-6A VICE1299a-6B V IC E 1557-19A VI C E0 86 6- 23 F V IC E 1066-N E S p VICE1485-19F VICE0869-19A V IC E 15 18 -6 A VICE1299b-14 VICE0902a-19F VICE1241a-11A VICE1260-6B V IC E 13 45 -6 A V IC E 1009-19A VICE1146-19F VIC E10 56 -2 3F VIC E09 24 b- 14 CI V E 08 3- 45 VICE1381-6B 3-6801 E CI V VICE1776-6B VICE1290-19A VICE1897-11A V IC E 1874b-N E S p VICE1575a-35B V IC E 0888-19A VI C E1 14 9a -2 3F VICE1865-15B/C V IC E 1845b-N E S p V IC E 1792-N E S p VICE0990b-14 71 E CI V F53-68 VIC E18 62 -1 0B VICE1877a-19F VICE1220-19F V IC E 1735-15B /C VICE1073-14 VICE1872-23B V IC E 09 56 -2 3F VI C E1 02 2- 18 C VIC E1409-23B F6 1- 73 11 E CI V V IC E 09 07 -6 C V IC E 0850-N E S p VIC E1093-N ESp VICE1313-19F VIC E09 77 -2 3F V IC E 1017-4 VICE1365b-NESp VICE0996-6B VIC E1764-11A VICE1036a-19A VICE1344a-11A VIC E1879a-6 B VICE1315-6A VIC E11 94 -2 3F VICE1452-21 VICE1395-19F VICE1461-19F VIC E1331-15B/C VICE1757-11A VICE1567-6C VIC E18 93 -1 0B V IC E 1727-N E S p V IC E 12 51 -6 A VICE0976-19A VICE1153-6B VIC E08 95 -1 8C B6-4990 E CI V VI C E1 08 9- 23 F V IC E 14 80 -2 3F VICE1807-35B VICE1573-6A V IC E 1087-N E S p VICE1473-6A VICE1058-6A VICE1580-14 VIC E1328a-9 V VICE1247-19F V IC E 0894a-9N VICE1486-21 VICE0948-6A VIC E1799-23B V IC E 1062-N E S p V IC E 11 89 a- 23 F A0 1- 48 80 E CI V VI C E1 46 0a -1 8C V IC E 1138-16F VICE0989a-6A VIC E1579-23B VICE1080-14 V IC E 1554a-9N VICE1378a-19A VIC E10 68 -1 9F V IC E 12 91 -2 3F VIC E1144-11A VICE1860-21 VICE0980-10A VICE1761b-33F VICE0892-6B VICE1244-14 V IC E 15 22 -2 3A VICE1125-6A VICE1270a-6B VIC E18 32 a- 10 B VICE1827-21 V IC E 13 25 -6 A V IC E 1296-9N VICE1861-6B VICE1079-14 V IC E 18 11 -1 9A V IC E 1798b-N E S p VICE1788-6B V IC E 11 35 -2 3F V IC E 0906-15B /C VICE1363-6B VICE1556-14 VICE1835a-33_Hybrid V IC E 14 92 -2 3A V IC E 1217-38 V IC E 1549b-38 VIC E1083-2 3F VICE1837-6C VICE1186-19A VICE1784-11A VIC E1479a-11A V IC E 1010-19A VICE1748-6C VICE1354a-11A V IC E 18 03 -2 2F VICE1262-15B/C V IC E 18 68 -6 A VICE1148-33F V IC E 15 27 -2 3F VICE1794-11A VI C E1 71 7- 22 F VICE0903-6A VICE1358-19F V IC E 15 32 -2 2F VI C E1 31 1- 23 F VICE1353b-14 VIC E10 16 -9 V V IC E 18 20 -2 3A V IC E 1236a-16F V IC E 08 76 -2 3F VICE1495-19A VICE0950-19A VICE1198-11A 3-1221 E CI V VI C E1 40 5- 23 F VICE1881-19F VICE1417-23B VIC E0855-15B/C VICE1413-6A V IC E 12 08 -6 A VICE1329-19F VICE1497-6A A6-4851 E CI V VICE1255b-19A VICE1099-6B VIC E11 79 -2 3A V IC E 09 27 -2 3F V IC E 1846-16F V IC E 1289-16F V IC E 13 21 -2 3F VICE1061-14 VIC E0904-N ESp V IC E 1726-15B /C V IC E 13 49 -6 A VICE1880b-19A VICE1078b-14 VICE0939-19F V IC E 14 01 -2 3A V IC E 11 11 -2 3F VICE1586-15B/C CI V 3- b8 33 1 E V IC E 1152-N E S p VICE1583-21 V IC E 18 71 -2 3F VIC E11 85 -2 3F VICE1549a-23B 3-6021 E CI V VICE1437-6B VICE1013-6A VICE1838-19A V IC E 1272-N E S p V IC E 14 93 -2 3A V IC E 1163a-3 VICE1060b-14 VICE1091-11A VIC E11 29 -2 3F VICE0919-19F V IC E 10 32 -2 3A VIC E0874-6B VICE0966-6B VICE1369-6A VICE0889-6A VICE1832b-14 VICE1088-19F VIC E11 54 -2 3F VICE1164-19F VICE1267-19A VICE0999-6B VICE1064-6A VICE1275b-14 V IC E 1162-3 VIC E1425a-23B V IC E 1572-N E S p V IC E 1280-N E S p VICE1420-23B V IC E 1770-9N VICE1362-6A V IC E 10 92 -2 3F VICE1005-11A VICE1435-35B VICE1072-11A V IC E 1797-21 VICE1214-19F VICE1424-6B VICE0951-19A V IC E 13 70 -2 3F VICE1416-6A VICE1336-15B/C V IC E 10 18 -6 A V IC E 18 64 -3V IC E 09 18 -6 C VICE1245-19F V IC E 13 88 -2 3F VICE1713-6A VICE1758-11A VICE1581-19A VIC E10 41 -2 3F VICE1839-11A V IC E 08 96 -2 3F VIC E0880-6Bii V IC E 09 61 -2 3F VICE1752-11A VIC E1158-11A VIC E1587-6Bii VI C E1 12 2- 23 F 3- 28 51 E CI V VICE1113-19A VIC E1870-11A V IC E 10 46 a- 23 A VI C E1 35 5- 23 F VICE0851-14 VICE0917a-19A VICE1057-6B V IC E 13 26 -6 A VIC E14 22 -1 8C V IC E 12 34 -3 VICE0993-6B VICE1389-19A V IC E 14 94 b- 23 A VICE1569-6C VI C E1 02 3- 18 C VICE1451-19F VICE1439-19A VICE1402-11A VICE0905-33F VIC E1312-11A V IC E 13 60 -3 VICE1105-14 VICE1744-19F VICE1365a-6B VICE1059-19F V IC E 14 32 -2 2F VIC E1795-23B VICE1160-6C VIC E0897-15B/C F53-9841 E CI V VICE0969-14 VIC E18 89 -1 0B VIC E1867-2 3B V IC E 13 08 -6 A VICE1127-6B A6-9980 E CI V F5 3- 23 71 E CI V VICE0974-6B VIC E18 33 -1 0B V IC E 1098-15B /C VICE1750-23B VICE1167-6B VICE1520-15B/C VICE1364a-6B VICE0933-6A VIC E15 70 -1 8C VICE1760-19F VICE1055-6A 3- 25 80 E CI V V IC E 18 49 -2 3A VICE1841-19A VICE1723-6B VICE1318-15B/C VIC E1490-6 B VICE1414-6B VICE1504-11A VIC E09 45 -9 V VI C E1 72 8- 22 F F5 3- 35 41 E CI V VICE0848-19F VIC E1045-2 3F VICE1816-6B V IC E 11 89 b- 3 A6-7780 E CI V VICE0978-11A V IC E 1874a-19A V IC E 1261a-38 V IC E 1806-16F VICE1777-14 VICE1044-6B V IC E 11 07 -2 3F VICE1115b-19A V IC E 11 17 -3 V IC E 1530-N E S p VICE1537-6B VIC E1712b-23B V IC E 1319b-N E S p VICE1768b-35B VIC E11 55 -2 3F VICE0944-6A VICE1440-35B VIC E11 93 -2 3F V IC E 1021-N E S p V IC E 15 58 -2 3A 3-9671 E CI V VICE0926-6B VICE1898-11A V IC E 1543-21 VIC E14 07 -2 3F V IC E 0920-35B 3-4190 E CI V VICE1133-6B VICE1028-19F V IC E 10 52 -3 VI C E1 34 4b -2 3F V IC E 1043-N E S p VICE1163b-6B V IC E 13 90 -2 3F V IC E 1563-15B /C V IC E 1712a-19A VICE1425b-33F VIC E1150a-6Bii V IC E 1053-35B V IC E 09 00 -2 3F VICE1235-19A V IC E 1000-N E S p VICE1076b-NESp VICE1001-6B VICE0873-23F VICE1773-15A VICE1857-19A V IC E 11 18 -2 3F VICE1347-19A VICE1885-19F VICE1284-19F VICE0911-33F VIC E1034-6Bii VICE1561-11A V IC E 10 19 -2 2F I V F5 3- 78 81 E C V IC E 09 71 -2 3F V IC E 1309b-N E S p VICE1853-33_Hybrid V IC E 0881-9N VI C E0 91 2a -2 3F VIC E08 86 -2 3A VI C E1 37 5- 23 F VICE1767-35B VICE1436-35B V IC E 13 98 -2 3A VICE1335-6A VIC E12 04 -2 3F VICE1140-19A V IC E 13 48 -6 A VICE1040-6B V IC E 15 59 -2 2F VICE1343a-15B/C V IC E 1257a-N E S p VICE1733-23B VIC E1230-15B/C VICE1566-6C VI C E1 17 2- 23 F F6 1- 17 01 E CI V VICE1033-14 VI C E1 13 1- 18 C VICE1259a-6B V IC E 18 63 -2 3A VI CE1 38 5- 18 C VICE1551a-15B/C V IC E 0997-N E S p VICE1285-19F VI C E1 72 9- 22 F VIC E1357-11A VICE1521-19F V IC E 15 33 -2 2F V IC E 1875-N E S p V IC E 1295-16F VICE0957-6B VICE1237a-19A VICE1145a-6A VICE1779b-14 VICE1394a-19F VICE1826-6B VICE1782-14 V IC E 14 55 -2 3A VICE1274-6B VIC E09 82 -2 3F 3- 18 01 E CI V VICE1412-6B V IC E 1265-38 V IC E 1286-19A V IC E 1406-19A VICE1100-11A VICE1283-19A V IC E 1320-N E S p VICE0924a-11A V IC E 1199b-N E S p VI C E1 88 2a -2 3F V IC E 1426-9N F6 1- b0 72 1 E CI V VICE1108-33F V IC E 09 83 -2 3A VIC E1895-15B/C VIC E12 13 a- 23 A VIC E1394b-15B/C VICE1063-11A VIC E1540-6Bii VIC E09 63 -2 3F VIC E1304-15B/C VICE0857-19A VIC E09 59 -9 V V IC E 12 29 -2 3F VICE1222-19A VICE1775-15B/C VICE0970-14 VICE1523-6A VI C E1 72 1- 22 F VIC E1282a-11A V IC E 0986-N E S p VICE1751-23B VICE1209-19A V IC E 1502-15B /C V IC E 0912b-15B /C VIC E1842-23B VIC E0968-9 V VICE1433a-23B VICE0988-15B/C VICE0849-6B V IC E 13 99 -2 3A 3-4811 E CI V VICE1828-11A VICE0943-6B VICE1139-19F V IC E 14 47 -2 3F VICE1524-19A VICE1097b-14 V IC E 1242a-16F VICE1128b-19F VICE1434-21 VIC E1411-15B/C VICE0952-6B V IC E 1734-15B /C V IC 3031 E -6 A VICE1848a-19A V IC E 1151-15B /C V IC E 09 55 -2 3F VICE1550-19A VIC E11 56 a- 23 F V IC E 13 82 -2 3F VICE1876-11A VICE1157-6B VICE1210a-19A VICE0940-6B VICE1288-19F VIC E10 49 -9 V V IC E 09 60 -2 3F VICE1337-11A V IC E 13 50 -6 A VICE1211-6A VIC E11 41 -1 4 VICE1415-11A VIC E1528-23B VIC E09 21 -1 8C VICE0908-33F VICE1014-6A VICE1171-6A VICE1060a-19F VICE1747-23B VICE1869-6A V IC E 1145b-16F VIC E1190-11A VICE1136-19A F5 3- 46 51 E CI V VIC E0870-11A VICE1494a-NESp VICE1818-6B 3- 86 21 E CI V V IC E 14 67 -2 3F VICE1218-33F V A6-7711 E CI VICE1501-15B/C VICE1740-35B VICE0887-6B VI C E1 17 8- 23 F V IC E 14 03 -2 3F VICE1884-19F VICE0942-6A VICE1456-6B VIC E10 96 -9 V VICE1143-19A VICE1446-35B V IC E 13 22 -2 3F V IC E 12 93 a- 23 F VI C E0 92 9- 18 C V IC E 13 27 -6 A VICE1106-6B V IC E 17 89 -3 V IC E 1553-9N 3-1211 E CI V V IC E 1386-N E S p VIC E1372-15B/C VICE1408-11A VIC E1541-23B VICE0964-19A 6131 E CI V 3- V IC E 1890-16F VICE1858-19A V IC E 12 54 -6 A 3- 56 90 E CI V V IC E 17 54 -6 C VICE1275a-21 VIC E09 58 -9 V VIC E1761a-23B V IC E 1149b-38 V IC E 1474-N E S p VICE0962-19F VICE1191-6A V IC E 17 83 -2 3A VICE1012-6B VICE1359-11A VICE1433b-35B VIC E1004-6Bii VICE1256-6A VICE1294-6A V IC E 17 42 -2 2F VICE1551b-19A VICE1714-23B A0 1- 24 41 E CI V VICE1483-19F V IC E 1287-9N V IC E 09 35 -2 3A V IC E 14 98 -3 V IC E 1258-N E S p V IC E 08 82 -3 VIC E1542-23B VICE1791a-11A V IC E 1090-N E S p VICE1429-6B VICE1821-6B VICE0947-19A VI C E1 34 2- 23 F 3-a8701 E CI V V IC E 1458-35B VIC E1183-6A VICE1488b-35B V IC E 1778-3 V IC E 1011b-19A V IC E 0937b-N E S p V IC E 1328b-N E S p VICE1896-6B VICE1845a-19A VICE1273-19F VICE0913-15B/C VICE1730-6B V IC E 18 59 -2 2F F5 3- 08 71 E CI V VICE0891-14 VICE0941-6B VIC E10 38 -2 3F VICE1200-11A VICE1393-14 V IC E 1848b-N E S p VICE1441-35B V IC E 1883a-N E S p VICE1736-21 VICE0879-14 VICE1110-19A V IC E 1297-16F V IC E 0858-3 V IC E 15 55 -2 2F VICE1377-6A VICE1589-6A V IC E 1856-19A VICE1085-19F VIC E1738-2 3B V IC E 1025b-9N VICE1763-33F V IC E 1379-38 VICE1843-19A VICE1886b-19F VICE1027-19F V IC E 14 81 -2 3F V IC E 17 45 -3 VICE1103-6B VICE1317-19F VICE1391-19A V IC E 12 52 a- 19 F VICE1340-6A 3-6451 E CI V 3- b6 61 1 E CI V VICE1292-19F VICE0990a-19F V IC E 1854-21 VICE1448-19F VICE1212-19F V IC E 10 42 -2 3A VICE0868-19A VICE1166a-19F VICE1739-19A VICE1484-19F VIC E1476-6B VICE1809-21 VICE1466-6A V IC E 13 23 -2 3F V IC E 09 15 -2 3F A0 1- 58 80 E CI V VICE0860-6B VICE1051-19F V IC E 1878-35B VICE1104-6B VICE1205-11A VIC E1159-15B/C V IC E 13 38 a- 23 F V IC E 1210b-N E S p VICE1577-6A VICE1774-15B/C VICE0938-19F VICE1517-15B/C VICE1423-6B VICE1835b-14 VI C E1 71 6- 22 F V IC E 1132-15B /C VIC E1150b-N ESp VICE1851-11A VICE1035-14 VI C E1 74 3- 22 F V IC E 1263-38 V IC E 1123-38 V IC E 15 91 -2 2F 3-6911 E CI V VICE1508-15B/C V IC E 13 71 -2 3F V IC E 18 88 -2 3F V IC E 18 14 -2 2F A6-0831 E CI V VIC E0872-11A VICE1030-6A VICE1850-11A VICE1077-6B VICE1421-23B VI C E1 58 5- 23 F V IC E 1084-N E S p V I 3-a5111 E C VIC E10 25 a- 23 F VICE1741-21 V IC E 1367-N E S p VI C E1 75 9a -2 3F 6-0341 E CI V A V IC E 14 72 -2 3F V IC E 1361-16F VIC E1471-15B/C VIC E1545-23B V IC E 1215-N E S p VICE1269-6C VICE1225a-19A V IC E 14 91 a- 23 A V IC E 1813-N E S p VICE1450-15B/C VIC E09 46 -1 4 V IC E 18 15 -2 2F V IC E 1293b-N E S p VICE1396-19F VIC E1790-23B VIC E1095-15B/C V IC E 1046b-38 V IC E 1460b-N E S p VIC E1588-6Bii VICE1496-15B/C VICE1192-15B/C V IC E 1240-19A VICE1050-19F V IC E 1791b-N E S p VICE1779a-15B/C VICE1834-33_Hybrid VIC E09 87 -2 3F 3- 61 21 E CI V VICE1202-11A VICE1161-19F VICE1525-35B V IC E 0916-15B /C V IC E 1771-9N V IC E 18 44 -2 3A VIC E1469-11A VIC E1428-15B/C V IC E 14 00 -2 3F F53-6251 E CI V VIC E13 06 -1 8C VICE0922-19A VIC E11 42 -1 4 V IC E 18 04 -2 2F VICE1054-19F V IC E 15 10 -2 3A V IC E 1462-19A VICE0863-6A V IC E 1352-N E S p VICE1459-14 A6-3831 E CI V VICE1276a-6B V IC E 11 88 -3 VICE1278-15B/C V IC E 1772-N E S p V IC E 1470-15B /C VICE0930a-6B VIC E1036b-2 3F VICE1102-6B A6-9281 E CI V VIC E09 17 b- 14 V IC E 17 87 -3 VICE1756-11A VICE1449-15B/C VICE1097a-6B V IC E 12 98 -3 VICE0985-6B VICE1147-6B -8421 E CI V 3 VICE1176-6B VICE1847-19A F53-1871 E CI V VIC E1246-6A VI C E1 71 8- 22 F VICE1109-6B VICE1410-6B V IC E 1227-38 V IC E 1310b-N E S p VIC E1187-11A I V 3-a8671 E C VICE1880a-6C V IC E 11 74 -3 V IC E 1354b-N E S p VIC E 0894b-N E S p VICE1444-21 VICE1438-19F VIC E11 95 -2 3F 3- 62 11 E CI V VICE1015-6A VICE1233-6B VIC E1307-15B/C VIC E09 95 -2 3F V IC E 15 31 -2 3F 3- 03 31 E CI V VICE1264-21 V IC E 13 66 -2 3F VICE1387-19A V IC E 1800-15B /C VICE1724-6A VICE1722-35B V IC E 1419-19A VI C E1 24 9- 18 C VIC E1562-15B /C VICE1341-6A VIC E0972-9 V VICE1305-19A VICE1067-14 VICE1503-19F VICE1332-6B VIC E18 30 -1 0B V IC E 11 30 -2 3F F6 1- 26 80 E CI V VICE1169-19A V IC E 12 76 b- 3 VICE1279-6B V IC E 1231-16F V IC E 1801-15B /C VI C E1 20 3- 23 F VICE1568-23B VIC E1310a-11A V IC E 0989b-N E S p VICE1065-14 V IC E 10 07 -6 A VICE1026-19F V IC E 13 53 a- 23 F V IC E 11 73 -2 3F V IC E 1266-38 VICE1397-19A VICE1238-14 VICE1253-6B V IC E 15 52 -6 A VIC E1812-15B/C V IC E 1746-N E S p VIC E1808-6Bii VICE0923-11A V IC E 09 91 a- 23 F V IC E 1571-N E S p VICE1817-6B VICE1753-11A V IC E 1343b-N E S p V IC E 14 31 -2 2F VICE1333-6A VICE1199a-11A V IC E 1500a-21 VICE1020-14 VICE1180-33F VICE0949-19F V IC E 11 20 -2 3F VICE1883b-19F VICE1368-6B F5 3- 04 81 E CI V VIC E1334-15B/C V IC E 15 05 -2 3F VICE1445-6A VICE1008-23B V IC E 1539b-N E S p VICE0973-19A VICE1755-11A V IC E 15 09 -2 3A VICE1810-6B V IC E 1877b-N E S p VICE1873-11A V IC E 1048-38 V IC E 17 93 -2 2F VI C E0 89 0- 18 CVI C E1 72 0a -2 2F VICE0867-19A V IC E 18 66 -2 3A VICE1574-15B/C VICE1487-19F VIC E10 94 -2 3A V IC E 1168-38 F6 1- 96 01 E CI V VICE0847-19F VICE1506-19F VICE1765-19F VICE1114-18C V IC E 0893-19A VICE1468-6B VICE1224-19A V IC E 14 88 a- 3 VI C E1 73 1- 22 F V IC E 15 34 -2 2F V IC E 1076a-N E S p VICE1519-15B/C VI C E1 53 5- 22 F VICE1170-6A VICE0954-14 A6-b7521 E CI V VICE1165-6B V IC E 1029-19A A0 1- 42 31 E CI V VICE1074-6A V IC E 1427-16F VICE1037-19A V IC E 1482-38 V IC E 17 49 -2 3A V IC E 0853-3 VICE1507-6A 3- 87 41 E CI V VIC E1836-23B V IC E 1891-16F V IC E 17 85 -3 V IC E 18 19 -2 3A VICE0865-19A VICE1075-11A V IC E 11 19 -2 3F 3-a6881 E CI V F5 VICE1134-19F VIC E09 67 -9 V VIC E09 84 -2 3F VICE1855-19A V IC E 1255a-38 VICE1477-19A VICE1404-6A V IC E 09 36 -3 1 VICE1499-15B/C A6-6351 E CI V VICE1082-6B V IC E 1003-N E S p VIC E15 90 -1 8C VICE1271-6B V IC E 0898-N E S p V IC E 0864-15B /C VICE1112-6B VICE1252b-19F V IC E 08 61 -2 3A VIC E18 92 -1 0B VICE1576-19A 3-6671 E CI V V IC E 0991b-N E S p V IC E 1715-19A VIC E12 39 a- 23 F VIC E1376-15B/C VIC E13 02 -1 8C VICE0975-19A VICE1116-18C VICE1175-19F VIC E1737-2 3B VICE0937a-6A VICE1464-6C V IC E 1798a-21 V IC E 1239b-16F VI C E0 91 0- 23 F VICE1250-11A VICE1894-6B VICE0878-23F VICE0934-6A V IC E 0992-N E S p VICE1762-21 VICE1047-6B 3- 42 01 E CI V VIC E0979-6Bii VICE1443-33F VI C E1 00 6- 23 F VI C E1 75 9b -2 2F VICE1539a-11A VICE1351-15B/C VICE1392-19A VICE0981-15B/C VICE1223a-6A V IC E 13 46 -6 A 3- b8 73 1 E CI V VICE1243-6A VIC E12 32 -1 9F VIC E10 70 -2 3F VICE0901-6B A6-4831 E CI V V IC E 15 60 -2 2F 3- b0 27 1 E CI V VICE0953-11A V IC E 13 56 -6 A V IC E 0909-N E S p VICE0871-19A VIC E1309a-11A V IC E 13 01 -2 3F VICE1339-15B/C V IC E 1039-38 VICE0859-6B VICE1002-6B V IC E 14 65 -2 3F VICE1802-15B/C V IC E 15 78 -2 3A V IC E 09 25 -2 3F VIC E1124-6A VICE1500b-15B/C 81 41 E CI V F5 3- VICE1031-14 V IC E 10 11 a- 22 F VIC E12 01 -2 3F V IC E 11 28 a- 23 A VICE1719-6B V IC E 09 31 -2 3F VICE1454-11A Tree scale: 0.001 Clonal complex CC236/271/320 CC439 CC199 CC138/176 CC62 CC180 CC460 CC490 CC124 CC392 CC433 CC344 CC448 CC177 CC1262 CC395 Other Tree annotation Serotype 19F 23F 6B 6A 19A 15B/C 11A 3 NESp 14 23A 23B 22F 6C 21 Other VT Other NVT Inner ring Study period PreVac PostVac Outer ring FIG 1 Phylogenetic tree created from 1,066 full-length coding loci found in 99.9% of 987 genomes from carriage samples and annotated with CC designations. Serotypes (inner circle) and study periods (outer circle) are also presented. Vaccination: Pneumococci from Carriage and Middle Ear Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 5 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://jcm.asm.org Serotype 6C belonged to CC3156B/C. Serotype 35B PNSP were only detected after vaccine introduction, and they were members of CC19835B, which was not previously detected in carriage (Table 2, Fig. 1, and Table S4). The proportion of MDR NESp isolates increased between the two study periods from 23.2% to 33.9% (P � 0.025) (Table 2), and 82.4% of MDR NESp PostVac isolates were members of CC344NT (Fig. 1 and Table S4). Middle ear samples from AOM. The Department of Clinical Microbiology received 6,651 ME samples during the study period. The total annual number of ME samples decreased from 966 samples in 2009 to 421 by the end of September 2017 (Table 3). Among all 6,651 samples, 994 were positive for pneumococci and 18 isolates were excluded, as they were not stored or not viable, leaving a total of 976 isolates for further analysis. The annual number of pneumococcal isolates from ME decreased from 197 (8.5/1,000 children aged 0 to �7 years) in 2009 to 44 (1.9/1,000 children) in 2016, and by the end of September 2017, the number of isolates was 23 (1.0/1,000 children) (Table 3). The median age of the children from which the ME isolates were obtained was 1.5 years, and 69.1% (n � 674) of the isolates were collected from the youngest age group (0 to �2 years of age). The average annual number of isolates in the youngest age group (0 to �2 years) decreased from 133.7 (18.1/1,000 children aged 0 to �2 years per year) PreVac to 45.5 (6.9/1,000 children aged 0 to �2 years per year) PostVac (P � 0.020) (see Table S5). The genomes of 441 (50.6%) pneumococcal isolates from 2009 to 2014 were sequenced. Serotypes. Overall, 894/976 (91.6%) pneumococcal isolates from ME samples were successfully serotyped, but 82 isolates (8.4%) were of serotypes other than those included in the mPCR scheme. Twenty-eight serotypes were detected overall: 23 PreVac and 22 PostVac. The numbers of VT pneumococci decreased significantly between the two periods (P � 0.001) (Table 3). The numbers of VT pneumococci decreased signif- icantly in the two younger age groups (children 0 to �2 years, P � 0.001; and 2 to �4 years of age, P � 0.005), while there was no change between the periods for the oldest age group (children 4 to �7 years of age; P � 0.450) (Table S5). The NVT pneumococci that increased PostVac in children aged 0 to �2 years were serotypes 15B/C (n � 1 to n � 40; P � 0.001), 6C (n � 1 to n � 29; P � 0.001), 23A (n � 2 to n � 18; P � 0.007), and 23B (n � 1 to n � 10; P � 0.042). Serotype 15B/C was the only NVT that increased in children 2 to �4 years of age (n � 1 to n � 11; P � 0.042), and serotype 6C (n � 12) was only detected PostVac within that age group (Table S5). MLST/CC. Among the 441 sequenced isolates, 41 CCs (29 CCs PreVac and 31 CCs PostVac) and 86 STs (55 STs PreVac and 52 STs PostVac) were detected, and 7 CCs and TABLE 2 Most common PNSP and MDR serotypes in nasopharyngeal samples PreVac (2009 to 2011) and PostVac (2012 to 2017) Serotype or sample type PNSP MDR Pna PreVac PostVac P value PreVac PostVac P valuen % n % n % n % 19F 74 49.7 59 17.5 �0.001 73 48.3 60 23.9 �0.001 NESpb 34 22.8 85 25.1 0.657 35 23.2 85 33.9 0.025 6B 12 8.1 4 1.2 �0.001 16 10.6 5 2.0 �0.001 14 11 7.4 1 0.3 �0.001 3 2.0 1 0.4 0.087 19A 6 4.0 35 10.4 0.021 3 2.0 9 3.6 0.516 6C 3 2.0 46 13.6 �0.001 4 2.6 47 18.7 �0.001 15A 0 0 29 8.6 �0.001 0 0 29 11.6 �0.001 23B 0 0 32 9.5 �0.001 0 0 1 0.4 1.000 35B 0 0 25 7.1 �0.001 0 0 0 0 NCc Other PNSP/MDRd 9 6.0 22 6.5 1.000 4 2.6 6 2.4 1.000 Total 149 338 0.269 151 251 0.030 aPn, pneumococci. bNESp, nonencapsulated S. pneumoniae. cNC, not calculated. dOther less prevalent serotypes of PNSP and MDR pneumococci. Quirk et al. Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 6 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://jcm.asm.org 24 STs were unique to ME isolates. The STs of two isolates could not be determined as a full-length allele was missing from the WGS data, but these isolates were members of CC180 and CC236/271/320. The Simpson diversity indices of the STs were 0.91 PreVac and 0.96 PostVac. A phylogenetic tree was created with the concatenated sequences of 1,250 full-length coding loci found in 99.8% of the ME pneumococcal genomes. The tree was annotated with CC designations and serotypes (Fig. 2). Fewer VT CCs were detected PostVac (6 CCs) than PreVac (18 CCs), and fewer STs were detected within the CCs. Six STs of VT isolates were detected within CC43923F PreVac, compared to only one ST PostVac (Fig. 2 and Table S6). CC236/271/32019F was the most common CC in both study periods: 41.8% (115/275, 4.8/1,000 children) PreVac, but it decreased in prevalence to 22.3% (37/166, 1.5/1,000 children) PostVac (P � 0.001) (Fig. 2 and Table S6). Between the periods, CC3156B/C increased from one serotype 6B isolate to six serotype 6C isolates (P � 0.05). CC126215B/C, CC19321, and CC181635B, which contained NVTs, were only detected PostVac in ME samples, but CC126215B/C was detected in low numbers in nasopharyngeal carriage PreVac. One isolate of NVT serotype 23B PostVac belonged to CC156/1629V, a typically VT lineage (Fig. 2 and Table S6). Antimicrobial resistance. The overall prevalence of PNSP among ME isolates decreased between the two study periods (48.1% versus 28.4%; P � 0.001). The TABLE 3 Serotype distribution each study year in ME samples among children 0 to �7 years old PreVac (2009 to 2011) and PostVac (2012 to 2017) Serotype or sample type No. of isolates 2009 to 2011 (avg/yr) No. of isolates PostVac 2012 to 2017 (avg/yr) P value2009 2010 2011 2012 2013 2014 2015 2016 2017a PreVac 3 3 2 7 3 9 4 0 0 0 12 4.0 16 2.7 0.635 4 1 0 0 0 0 0 0 0 0 1 0.3 0 0 NCb 6A 14 22 11 13 12 2 1 1 0 47 15.7 29 4.8 0.159 6B 17 14 6 8 2 1 0 0 0 37 12.3 11 1.8 0.010 6C 0 0 1 2 10 10 7 8 4 1 0.3 41 6.8 �0.001 9N 0 0 1 0 0 0 0 0 0 1 0.3 0 0 NC 9V 4 2 1 0 0 0 0 0 0 7 2.3 0 0 NC 10B 0 0 0 0 1 0 0 0 0 0 0 1 0.2 NC 11A 1 4 3 6 3 4 1 2 1 8 2.7 17 2.8 0.239 14 14 13 10 1 4 1 0 0 0 37 12.3 6 1.0 �0.001 15A 0 0 0 0 1 1 0 1 3 0 0 6 1.0 NC 15B/C 2 2 1 15 22 2 5 8 1 5 1.7 53 8.8 �0.001 16F 1 1 0 0 0 0 0 1 0 2 0.7 1 0.2 0.695 17 0 1 0 0 0 0 0 0 0 1 0.3 0 0 NC 18C 1 1 0 1 0 0 0 0 0 2 0.7 1 0.2 0.695 19F 85 69 88 33 27 5 0 2 0 242 80.7 67 11.2 �0.001 19A 17 13 5 8 6 5 1 1 1 35 11.7 22 3.7 0.240 19C 1 0 0 0 0 0 0 0 0 1 0.3 0 0 NC 21 0 0 0 3 5 1 1 2 1 0 0 13 2.2 NC 22F 0 0 0 1 1 2 0 0 0 0 0 4 0.7 NC 23F 29 21 23 9 6 3 0 0 0 73 24.3 18 3.0 �0.001 23A 1 1 0 2 5 1 8 6 1 2 0.7 23 3.8 0.003 23B 0 0 1 5 3 2 1 2 2 1 0.3 15 2.5 0.012 24F 0 0 1 0 0 0 0 2 0 1 0.3 2 0.3 0.707 33F 0 0 4 3 4 3 0 1 0 4 1.3 11 1.8 0.231 35F 0 1 0 0 3 0 1 0 0 1 0.3 4 0.7 0.365 35B 0 0 0 3 2 2 1 1 0 0 0 9 1.5 NC 38 1 0 1 0 0 0 0 0 0 2 0.7 0 0 NC Other serotypesc 4 10 5 13 12 11 11 6 9 19 6.3 62 10.3 0.001 NESpd 1 0 0 1 0 0 0 0 0 1 0.3 1 0.2 0.995 Total 197 177 169 130 138 60 38 44 23 543 181 433 72.2 0.014 VTe 151 120 128 52 39 10 0 2 0 399 133.0 96 16.0 �0.001 NVTf 46 57 41 78 99 50 38 42 23 144 49.0 337 56.2 �0.001 All ME samples 966 926 951 849 894 687 505 452 421 2,843 947.7 3,808 634.7 Samples positive for Png (%) 20.4 19.1 17.8 15.3 15.4 8.7 7.5 9.7 5.5 19.1 11.4 aFrom 1 January to 30 September 2017. bNC, not calculated. cSerotypes other than those included in the multiplex PCR panel of the study. dNESp, nonencapsulated S. pneumoniae. eSerotypes detected in the study that are included in PHiD-CV (4, 6B, 9V, 14, 18C, 19F, and 23F). fSerotypes detected in the study that are not included in PHiD-CV. gPn, pneumococci. Vaccination: Pneumococci from Carriage and Middle Ear Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 7 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://jcm.asm.org resistance to erythromycin decreased between the two periods (49.7% versus 29.8% of isolates; P � 0.001), and the prevalence of MDR pneumococci decreased also (49.2% versus 29.8%; P � 0.001). Before the introduction of PHiD-CV, 98.1% (56/261) of PNSP were also MDR, and this was reduced to 91.9% (113/123; P � 0.001). PNSP and MDR isolates were mainly serotype 19F in both study periods (Table 4). Among the PNSP serotype 19F isolates, 89.2% belonged to CC236/271/32019F (Fig. 2 and Table S6). NVT VICE0354-6A VICE0507-11A VICE0338-23F VI C E0 32 8- 9V F3 2- 72 30 E CI V V IC E 05 66 -1 9F VICE0471-19A VICE1935-15B/C VICE0617-3 V IC E 03 06 -1 9F VICE1631-19A VICE0565-19F VIC E0438-19F VICE0404-19F VIC E0489-1 9F VICE0527-9N VIC E0595-6A VICE0472-19A V IC E 0446-14 VICE0608-6A VIC E0363-19F F3 2- 24 30 E CI V V IC E 06 27 -2 3F VI C E1 61 7- 19 F VICE0510-23F VICE0315-19A VICE1658-6C VICE0384-6A VICE0064-19F VICE0469-14 V IC E 0334-19F VI C E0 28 9- 16 F V IC E 04 23 -2 3F VICE0288-6Bii VIC E1923-11A VIC E1924a-11A V IC E 03 23 -2 3F V IC E 0589-19F VICE0519-23F VICE0027-19F VICE0320-3 V IC E 05 55 -2 3F VICE0415-6B V IC E 04 45 -2 3F V IC E 0452-14 VICE1647-6A VIC E0396-19F VICE0070-19F V IC E 03 49 -1 9F V IC E 03 87 -2 3F VI C E0 51 5- 19 F VI C E0 51 4- 19 F VIC E19 47 -2 2F VICE0358-19A VI CE0 48 4- 19 F VICE0607-15B/C V IC E 16 46 -1 9F VICE0035-19F V IC E 05 54 -2 3F V IC E 0625-19F V IC E 0493-19F VICE0324-6B V IC E 0353-19F VICE1936-19A V IC E 03 09 -1 9F VI C E0 53 7- 14 V IC E 04 75 -2 3F F91-3850 E CI V VICE1929-35B VIC E0616-11A V IC E 16 09 -2 3F VIC E05 33 -1 9F V IC E 05 57 -1 9F VICE0456-3 VICE0460-6A V IC E 04 77 -2 3F VICE1606-15B/C VIC E1943-6A VICE1664-19A VICE0382-6A VICE0447-6Bii VICE0619-21 V IC E 0482-19F VICE1662-21 VICE0386-14 VICE1953-19A VICE1941-35B VICE1656-33F V IC E 0011-19F VIC E0398-6A VICE0464-19F VICE0612-15B/C VIC E0582-19F V IC E 05 86 -1 9F V IC E 1952-19F VICE0419-19F B32-2491 E CI V VICE0590-6A VICE0394-6A V IC E 06 04 -2 3F V IC E 19 44 -1 9F VICE0573-19A V IC E 0442-19F V IC E 02 82 -1 9F V IC E 0521-14 VICE0369-23F VIC E0352-6A V IC E 04 27 -1 9F VICE0609-35B VICE0497-19F VICE0308-6A V IC E 16 35 -1 9F V IC E 0468-19F VICE0383-19F V IC E 04 55 -1 9F VIC E0281-11A V IC E 0318-19F VICE0304-19F F9 1- 12 00 E CI V VICE1925-33F V IC E 03 10 -1 9F F91-5430 E CI V VIC E0433-6A VI C E1 67 2- 23 B VICE0388-14 F9 1- 41 61 E CI V V IC E 0574-6C VICE0563-19F VICE1937-19A VICE0293-6Bii V IC E 0301-19F V IC E 0389-19F V IC E 03 25 -2 3F VIC E16 28 -6 C V IC E 1983-6C VICE0520-19F VIC E05 50 -6 C VICE0621-NESp V IC E 04 01 -2 3F VICE0375-6B VICE0488-23F VICE0478-6A F3 2- 38 20 E CI V VICE0592-6A VICE0332-19A VICE0311-6A V IC E 0457-19F VI C E0 37 0- 19 F VIC E0287-6 A VICE0556-33F VICE0337-6A V IC E 0392-19F V IC E 05 91 -2 3F VIC E0529-19F VICE0336-19A VICE0420-6B VICE0540-11A VICE0290-19A VICE1619-14 VIC E03 44 -1 9F VICE0584-6A VIC E1621-19F V IC E 04 76 -1 9F VICE0461-14 V IC E 0374-23F VIC E0571-19F VICE0393-6A VICE0479-19F VICE0549-3 V IC E 0546-19F V IC E 03 62 -2 3F VI C E0 45 9- 16 F VICE1949-19A VICE1660-21 VICE1633-15B/C VIC E16 55 -3 5F V IC E 00 20 -1 9F V IC E 04 32 -1 9F VIC E1955-1 9F VICE1926-3 V IC E 0580-19F B32-9261 E CI V V IC E 03 59 -2 3F VI C E0 50 3- 38 V IC E 1641-19F VICE0487-19F VICE0428-6B VICE0385-23F V IC E 0597-19A VICE0314-19F VICE0347-19A F91-6340 E CI V VIC E05 22 -1 9F VICE1659-15B/C VICE1625-19A VICE0378-19F VICE1622-3 VICE0312-14 VICE0467-19F V IC E 0496-19F V IC E 03 65 -1 9F VICE1634-6A VIC E0351-6A VIC E0340-6A VICE0577-6B VIC E05 35 -1 9F V IC E 03 76 -2 3F VICE0567-6B V IC E 06 18 -1 9F V IC E 03 55 -2 3F VICE1668-6A VIC E16 24 -3 5F VICE0317-6Bii VIC E1607-19F VICE1650-14 VICE0360-6B V IC E 0426-14 VICE0600-15B/C VICE0343-19F VICE0513-19F F3 2- 12 30 E CI V V IC E 1623-19F VICE0463-19F VICE0300-19F F91-9730 E CI V VICE0603-11A V IC E 05 62 -2 3F VICE1666-15B/C VICE0576-33F V IC E 1954-23A V IC E 19 33 -1 9F V IC E 03 67 -9 V VICE0569-19A VICE0372-19F V IC E 1661-23A V IC E 0564-19F VICE0596-11A V IC E 0498-19F VICE1928-15B/C V IC E 05 47 -2 3F V IC E 05 58 -1 9F VICE0543-3 VICE1948-15A VICE0402-6A V IC E 03 03 -2 3F V IC E 04 85 -1 9F VICE0437-6B V IC E 0413-19F V IC E 00 02 -1 9F V IC E 05 99 -1 9F F9 1- 58 50 E CI V V IC E 05 18 -1 9F VICE1932-23B VICE0501-14 VICE0458-6B V IC E 0036-19F V IC E 16 39 -2 3F V IC E 04 09 -2 3F VICE0528-19A VICE0579-35B V IC E 03 31 -1 9F V IC E 03 30 -2 3F V IC E 0453-19F V IC E 16 13 -1 9F VIC E0417-19F VICE0397-6B VICE0615-11A VICE0525-33F V IC E 06 26 -2 3F V IC E 16 11 -1 9F VICE0026a-19F VI C E0 54 4- 19 F V IC E 03 68 -2 3F VICE0568-19F VICE0570-14 F3 2- 41 40 E CI V VICE1946-11A VICE0508-6B VIC E0335-19F VICE1645-21 VICE0435-19F VICE0509-6Bii VICE1939-3 V IC E 0572-19F VICE0473-19F V IC E 1626-6C V IC E 06 02 -2 3F VIC E03 41 -1 9F F9 1- 38 40 E CI V VICE0366-14 VICE0407-19A V IC E 0072-19F V IC E 0400-19F VICE1604-33F V IC E 06 24 -1 9F V IC E 04 41 -9 V V IC E 0551-19A VICE0434-19A VICE0377-19F VICE0561-3 VICE0594-19A VICE0451-19A VICE1615-15B/C V IC E 1649-23A F9 1- 35 61 E CI V VICE0450-23F VICE0560-24F F32-4940 E CI V VIC E06 10 -2 2F VICE0593-6B V IC E 0292-19F V IC E 1627-23A VICE1648-6A VIC E0280-19F V IC E 05 11 -2 3F VICE0291-3 VICE0532-19F VICE0623-6A VICE0470-6A VICE0390-6A B32-7850 E CI V V IC E 0502-14 VIC E16 69 -1 9F VICE0416-19A V IC E 0449-19F VICE0444-14 V IC E 16 40 -1 0B VICE0622-6B VICE0512-19F VIC E0559-19F VIC E0506-1 9F VICE1654-19A VICE0294-19A V IC E 05 04 -1 9F VIC E05 05 -1 9F VICE0299-6Bii V IC E 0524-19F VICE1618-15B/C V IC E 0285-19F V IC E 0346-23A VICE1612-15B/C VICE0439-6B V IC E 0545-19F VIC E16 70 -6 A VI C E0 35 7- 18 C VICE0495-14 VICE0443-19A V IC E 1657-19A VIC E04 22 -3 5F V IC E 1938-6C VICE0326-6B V IC E 05 26 -9 V VICE0411-23F VICE0406-19F VICE0605-3 VICE1644-15B/C VIC E0531-6Bii VICE0297-19A V IC E 0578-19F B32-0260 E CI V VICE0490-15B/C VICE0538-19F VICE1636-6A VICE1637-15B/C V IC E 0541-19F VICE1674-35B VICE0462-15B/C VICE0553-14 V IC E 0356-19F VIC E0399-6A V IC E 04 30 -1 9F V IC E 1930-6C F91-8430 E CI V VICE1934-19A VIC E02 98 -1 9F VICE1610-3 V IC E 0284-19F VIC E02 86 -1 9F VI C E0 07 6- 19 F V IC E 0448-19F VI C E0 54 8- 19 F VICE0581-6B V IC E 16 65 -2 3A V IC E 0523-19F VICE0465-6A V IC E 0614-21 VICE0613-19A VICE0395-19A VIC E19 51 -6 C V IC E 03 91 -9 V V IC E 04 74 -1 9F V IC E 1675-6C VICE0500-6A VIC E03 19 -1 9F VICE0418-23F V IC E 1616-19F VICE0588-19FVICE0302-19F F91-3300 E CI V VIC E16 32 -6 C VICE0486-3 F9 1- 57 50 E CI V V IC E 03 33 -1 9F V IC E 0517-19F VICE1651a-11A VI CE0 44 0- 18 C V IC E 0491-19F VICE0601-21 B32-3761 E CI V V IC E 1605-23A V IC E 1630-14 VICE0361-15B/C VICE1642-15A VICE0412-11A VICE0380-14 VIC E1608-19F VIC E0492-6A VICE1671-15B/C VICE1663-6A V IC E 03 29 -1 9F VICE0408-19A VIC E0313-19F VICE1931-33F VICE0552-3 VICE0381-19A VIC E0403-19F VIC E16 76 -2 2F VIC E19 50 -2 2F F3 2- 46 30 E CI V V IC E 16 38 a- 19 F VICE0606-3 VI C E0 42 4- 19 F VIC E03 71 -1 9F VICE0534-19F VIC E16 43 -1 9F VI C E0 43 1- 14 VIC E1667-3 5F VICE0454-6B V IC E 0466-19F V IC E 0295-6B ii V IC E 0429-19F Tree scale: 0.001 Clonal complex CC236/271/320 CC439 CC199 CC138/176 CC62 CC180 CC460 CC490 CC124 CC392 CC433 CC177 CC1262 CC395 Other Tree annotation Serotype 19F 23F 6B 6A 19A 15B/C 11A 3 NESp 14 23A 23B 22F 6C 21 Other VT Other NVT Inner ring Study period PreVac PostVac Outer ring FIG 2 Phylogenetic tree created from 1,250 full-length coding loci found in 99.8% of the 441 genomes from ME samples and annotated with CC designations. Serotypes (inner circle) and the study periods (outer circle) are also presented. TABLE 4 Most common PNSP and MDR serotypes in ME samples PreVac (2009 to 2011) and PostVac (2012 to 2017) Serotype PNSP MDR Pna PreVac PostVac P value PreVac PostVac P valuen % n % n % n % 19F 231 88.6 66 53.7 �0.001 232 86.9 65 50.4 �0.001 6B 9 3.4 3 2.4 0.759 16 6.0 3 2.3 0.017 14 6 2.3 2 1.6 1.000 5 1.9 2 1.6 0.472 19A 4 1.5 9 7.3 0.006 2 0.7 9 7.0 0.014 23F 4 1.5 0 0 0.311 4 1.5 0 0 0.134 6C 0 0 19 15.4 �0.001 0 0 30 23.3 �0.001 15A 0 0 6 4.9 0.001 0 0 6 4.7 0.007 23B 1 0.4 5 4.1 0.014 1 0.4 1 0.8 1.000 Other PNSP/MDR Pnb 6 2.3 13 10.6 0.001 7 2.6 13 10.1 0.040 Total 261 123 �0.001 267 129 �0.001 aPn, pneumococci. bOther less prevalent serotypes of PNSP and MDR pneumococci. Quirk et al. Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 8 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://jcm.asm.org serotype 6C amounted to 15.4% of PNSP (P � 0.001) and 23.3% of MDR pneumococci (P � 0.001) detected PostVac (Table 4). The serotype 6C PNSP/MDR isolates were not detected before vaccine implementation and were members of CC3156B,6C. Serotype 19A PNSP increased from 1.5% to 7.3% (P � 0.006), and MDR serotype 19A increased from 0.7% to 7.0% (P � 0.014) (Table 4). Serotype 19A PNSP/MDR pneumo- cocci were members of various CCs (Fig. 2 and Table S6). NVT PNSP with serotypes 15A and 23B also increased after vaccine introduction, and they were members of CC6315A and CC33823B, respectively. NVT serotype 15A PNSP (also MDR) was only detected after vaccination in ME isolates (Table 4, Fig. 2, and Table S6). Comparison of serotypes in nasopharyngeal isolates from carriage and ME isolates from children with AOM 1 to <4 years of age. Overall, 2,291 pneumococcal isolates were obtained from children 1 to �4 years of age: 1,457 isolates from nasopharyngeal samples from carriage (n � 493 PreVac and n � 964 PostVac) and 834 isolates from AOM ME samples (n � 467 PreVac and n � 367 PostVac). The same serotypes were among the most prevalent serotypes PreVac in both sample groups, and the levels of serotype replacement PostVac were similar in both groups (Table 5, Fig. 3). One isolate of VT serotype 23F was detected in carriage among children 1 to �4 years of age in 2017, and no VTs were detected in children with AOM within the same age group after 2016. Serotype 6A decreased slightly PostVac among children with AOM but not in carriage (P � 0.050) (Fig. 3). Serotypes 6C, 15B/C, 23A, and 23B increased (Fig. 3). VT serotype 19F was more frequently found in AOM than in carriage in children 1 to �4 years of age in both study periods: 32.1% (268/834) of ME isolates were serotype 19F compared to 7.2% (105/1,457) of carriage isolates (P � 0.001). The NVT serotype 23B was more frequently found in carriage than in AOM in children 1 to �4 years of age: 5.0% (73/1,457) of carriage isolates were serotype 23B compared to only 1.7% (14/834) of the ME isolates (P � 0.006). Serotype 6C was prevalent in both carriage and AOM among children 1 to �4 years of age, but MDR was more common in ME isolates (P � 0.001). NESp isolates were more frequently found in carriage than among children with AOM in both study periods (P � 0.001) (Table 5). TABLE 5 The most common VTs and NVTs in carriage and ME samples from children 1 to �4 years old PreVac (2009 to 2011) and PostVac (2012 to 2017) Serotype or sample type PreVac PostVac Carriage ME P value Carriage ME P value No. of isolates % positive cultures No. of isolates % positive cultures No. of isolates % positive cultures No. of isolates % positive cultures 6A 48 9.7 43 9.2 0.766 76 7.9 28 7.6 0.889 6B 63 12.8 33 7.1 0.003 37 3.8 10 2.7 0.332 6C 6 1.2 0 0 0.018 90 9.3 36 9.8 0.696 11A 25 5.1 6 1.3 �0.001 64 6.6 15 4.1 0.074 15B/C 13 2.6 2 0.4 0.005 88 9.1 47 12.8 0.051 19F 63 12.8 215 46.0 �0.001 43 4.5 53 14.4 �0.001 19A 44 8.9 27 5.8 0.061 67 7.0 17 4.6 0.117 23F 67 13.6 65 13.9 0.830 45 4.7 17 4.6 0.993 23A 16 3.2 1 0.2 �0.001 70 7.3 19 5.2 0.172 23B 1 0.2 1 0.2 0.975 72 7.5 13 3.5 0.006 Less prevalent serotypes 111 22.5 73 15.6 NCa 255 26.5 111 30.2 NC NESpb 36 7.3 1 0.2 �0.001 59 6.1 1 0.3 �0.001 Total 493 100 467 100 NC 964 100 367 100 NC VTc 256 51.9 353 75.6 �0.001 141 14.6 87 23.7 �0.001 NVTd 237 48.1 114 24.4 �0.001 823 85.4 280 76.3 �0.001 aNC, not calculated. bNESp, nonencapsulated S. pneumoniae. cSerotypes detected in the study that are included in PHiD-CV (4, 6B, 9V, 14, 18C, 19F, and 23F). dSerotypes detected in the study that are not included in PHiD-CV. Vaccination: Pneumococci from Carriage and Middle Ear Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 9 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://jcm.asm.org DISCUSSION This study shows a significant reduction of VTs in nasopharyngeal carriage of healthy children and in ME samples in children �7 years old 6 years after PHiD-CV implementation in Iceland. The total number of pneumococci isolated from the naso- pharynges of children remained unchanged after vaccination due to serotype replace- ment by NVTs; however, at the same time, the total number of ME isolates decreased significantly. The ME samples in our study were most often from children with ruptured tympanic membranes as a consequence of AOM or from children with tympanic tubes and discharge from the middle ear as a consequence of inflammation in the middle ear. Consequently, we postulate that the decrease in the number of samples from the ME most likely reflects a decrease in the burden of AOM (52). Certain serotypes are associated with nasopharyngeal carriage in healthy children, while others are more prone to cause disease (53, 54); however, the NVTs that have replaced the VTs following routine vaccination may, to a great extent, express a low invasive disease potential (55). It was confirmed in our study that the distribution of serotypes and genetic lineages in nasopharyngeal carriage in children reflected those identified from the discharges from ears of children with AOM (56). Furthermore, the diversity of the pneumococcal STs did not change between the periods. The same serotypes and genetic lineages were often found in nasopharyngeal and ME samples in children 1 to �4 years of age, both PreVac and PostVac but often in different proportions. There was a slight but significant decrease in serotype 6A among children with AOM, which has been described in IPD among all ages following PHiD-CV vaccination (57). Serotypes 6C, 23A, and 23B were not affected by the vaccination, as these serotypes increased significantly in both sample groups, which has also been documented elsewhere (21, 58–62). Serotype 19F was particularly “otogenic,” as it was four times more prevalent in ME samples than in nasopharyngeal samples. The main serotype 19F in CC236/270/32019F harbors genes for pili (both PI-1 and PI-2), which likely contributed to the adherence to the mucosa of the middle ear (63). Serotype 6B had a preference for nasopharyngeal FIG 3 The annual average numbers of the most prevalent serotypes and NESp isolates detected in carriage versus ME samples among children 1 �4 years of age PreVac (2009 to 2011) and PostVac (2012 to 2017). �, decreased significantly between the two study periods; *, increased significantly between the two study periods; Nc, not calculated; NESp, nonencapsulated S. pneumoniae. Quirk et al. Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 10 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://jcm.asm.org colonization PreVac, while the same was true for serotype 23B PostVac. This finding is in concordance with other reports where serotype 23B was associated with persistent carriage and posed a lower risk for IPD (55, 64). The NESp isolates were largely associated with carriage in both study periods but extremely rare in AOM. However, while the prevalence of NESp did not change significantly after vaccination in carriage, MDR among NESp isolates increased signifi- cantly. In our study, only one VT isolate was detected between 2014 and 2016 in ME samples among children 1 to �4 years of age, whereas 11 VT isolates were detected between 2014 and 2017 in the nasopharynges of healthy children of the same ages. This indicates that PHiD-CV may be more effective in preventing AOM (measured as fewer positive ME samples) than in preventing nasopharyngeal carriage. This is impor- tant, as AOM caused by pneumococci is more severe than that caused by other common pathogens (65, 66). The increase of the MDR serotype 6C isolates PostVac in both nasopharynx and ME samples is of concern. Other researchers have also described an increase in serotype 6C following the introduction of PCV7, especially in non-IPD among children (67). We rarely detected isolates of serotype 6C PreVac; however, in a recent study, serotype 6C was only detected among vaccinated children with non-IPD after PHiD-CV implemen- tation (68). Serotype 6C isolates detected PostVac most often belonged to CC3156B and ST3866C (a double locus variant [DLV] of PMEN Poland6B-20). The MDR ST3866C was detected in Spain 6 years after PCV7 implementation (69), and other countries have also reported the emergence of this lineage in nasopharyngeal carriage and IPD following the implementation of PHiD-CV (62). This lineage might be derived from a capsular switch from serotype 6B to serotype 6C, as has previously been reported by our group (36). The majority of serotype 23B isolates belonged to CC439/ST43923B (single locus variant [SLV] of PMEN Tennessee23F-4); however, this lineage was only detected PostVac in both sample groups in our study. CC439/ST43923B was present, although uncom- mon, in Germany prior to the implementation of vaccinations but increased after vaccine implementation. This lineage has also been documented in other countries worldwide (70). One ME isolate of serotype 23B belonged to CC156/162 and ST162 (an SLV of PMEN Spain9V-3). This was the only isolate of this lineage detected PostVac in ME isolates. Serotype switch variants of the related ST156 expressing serotypes 9V, 9A, 14, 19F, and 11A have also been reported (71). Pneumococci have the ability to change their capsular serotype by exchanging the capsular locus genes (72). The expansion of preexisting lineages and their variants, such as CC3156B/C and CC43923F/B, may be more likely following PCV vaccination than the emergence of new lineages (70). Iceland offers a unique opportunity for researching vaccine effects for several reasons. The reference laboratory at the Department of Clinical Microbiology, Landspi- tali University Hospital, serves approximately 85% of the country for pneumococcus- positive samples and stores all isolates (at �80°C). Furthermore, carriage studies have been conducted within the same DCCs using the same methodology throughout the study period. PCVs were not part of the routine infant immunization program before PHiD-CV implementation in 2011. Since then, vaccine acceptance and the uptake of PCVs have been high (73, 74). In our study, we analyzed a large number of pneumo- coccal isolates representative of the Icelandic population. Furthermore, one third of the pneumococcal isolates were subjected to WGS, which gives a good overview of the composition of genetic lineages in the country. However, fluctuations in serotypes and genotypes are known among pneumococci, even without the selective pressure of PCVs (75, 76). In conclusion, PHiD-CV implementation eliminated VTs in the MEs of children with otitis media within 5 years. The carriage rate of pneumococci in healthy children remained constant between the periods due to serotype replacement of NVTs, but the total number of ME isolates decreased significantly PostVac. Serotype 23B and NESp Vaccination: Pneumococci from Carriage and Middle Ear Journal of Clinical Microbiology December 2018 Volume 56 Issue 12 e01046-18 jcm.asm.org 11 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/j cm o n 08 D ec em be r 20 24 b y 20 01 :4 53 0: 2: 20 3: ff ff :f ff f: ff ff :f fe 2. https://jcm.asm.org had a preference for nasopharyngeal carriage among children 1 to �4 years of age. Multidrug resistance among serotype 6C was more common in ME samples among children 1 to �4 years of age than in nasopharyngeal carriage samples PostVac. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/JCM .01046-18. SUPPLEMENTAL FILE 1, PDF file, 0.1 MB. SUPPLEMENTAL FILE 2, PDF file, 0.1 MB. SUPPLEMENTAL FILE 3, PDF file, 0.1 MB. SUPPLEMENTAL FILE 4, PDF file, 0.1 MB. SUPPLEMENTAL FILE 5, PDF file, 0.1 MB. SUPPLEMENTAL FILE 6, PDF file, 0.1 MB. ACKNOWLEDGMENTS This was an investigator-initiated study funded by GlaxoSmithKline Biologicals SA. Grants were received from the Landspitali University Hospital Research Fund, the Eimskip University Fund, the Wellcome Trust (research fellowship 083511/Z/07/Z and grant 04992/Z/14/Z to A.B.B.), and the John Fell Fund (grant 123/734 to A.B.B.). Work at the Wellcome Sanger Institute was supported by the Wellcome core funding (grant 206194 to S.D.B.). GlaxoSmithKline Biologicals SA was provided the opportunity to review a draft version of the manuscript, but the authors are solely responsible for the final content and interpretation. The authors received no financial support or other form of com- pensation related to the development of the manuscript. We thank the staff at the Department of Clinical Microbiology for collecting the isolates from ME samples. We also thank the teachers and staff at the DCCs and the children participating in the carriage studies and their parents. We also thank other members of the VIce study group. REFERENCES 1. Quintero B, Araque M, van der Gaast-de Jongh C, Escalona F, Correa M, Morillo-Puente S, Vielma S, Hermans PW. 2011. Epidemiology of Strep- tococcus pneumoniae and Staphylococcus aureus colonization in healthy Venezuelan children. Eur J Clin Microbiol Infect Dis 30:7–19. https://doi .org/10.1007/s10096-010-1044-6. 2. Bogaert D, De Groot R, Hermans PW. 2004. 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Middle ear study and bacterial isolates. Serotyping. DNA extraction and whole-genome sequencing. Genomic analyses. Antibiotic susceptibility testing. Statistical analyses. Ethics. RESULTS Nasopharyngeal samples from children attending DCCs. Serotypes. MLST/CC. Antimicrobial resistance. Middle ear samples from AOM. Serotypes. MLST/CC. Antimicrobial resistance. Comparison of serotypes in nasopharyngeal isolates from carriage and ME isolates from children with AOM 1 to <4 years of age. DISCUSSION SUPPLEMENTAL MATERIAL ACKNOWLEDGMENTS REFERENCES