Molecular characterization of Streptococcus pneumoniae, particularly serotype19A/ST320, which emerged in Krasnoyarsk, Russia.

Streptococcus pneumoniae, a common human pathogen, colonizes the nasopharynx and causes diseases including acute otitis media (AOM). Herein, pneumococcal serotype distributions in children before and after PCV7 vaccination and in patients with pneumococcal disease in Siberian Russia (Krasnoyarsk) are reported. Analyses included antimicrobial susceptibility testing, sequence typing (ST), pulsed field gel electrophoresis, virulence-related surface protein gene (VSG) typing with novel primers and structural analysis by scanning electron microscopy. In healthy children (HC) prior to administration of PCV7, drug-susceptible serotype23F/ST1500 was a major pneumococcal genotype. In the PCV7 trial, multidrug-resistant serotype19A/ST320 emerged in vaccinees after PCV7, exhibiting a PCV7-induced serotype replacement. Multidrug-resistant serotype19A/ST320 was evident in patients with AOM. Community-acquired pneumonia (CAP) isolates showed genetic similarities to the AOM (ST320) genotype, constituting a common non-invasive AOM-CAP group. In contrast, meningitis isolates were more divergent. Overall, 25 ST types were identified; five (20%) of which were Krasnoyarsk-native. Regarding VSGs, PI-1 (rlrA/rrgB), PI-2 (pitA/B), psrP and cbpA were present at 54.3%, 38.6%, 48.6%, and 95.7%, respectively, with two major VSG content types, PI-1- /PI-2- /psrP+ /cbpA+ and PI-1+ /PI-2+ /psrP- /cbpA+ , being found for HC and non-invasive diseases, respectively. A major clone of serotype19A/ST320 (PI-1+ /PI-2+ ) produced the longest pneumococcal wire (pilus) structures in colonies. ST1016 (PI-1- /PI-2- ) in HC had HEp-2 cell-adherent pili. These results suggest that serotype19A/ST320 and related genotypes, with the VSG content type PI-1+ /PI-2+ /psrP- /cbpA+ , emerged in vaccinees after PCV7 in Siberia, accompanying diseases in non-vaccinated children, and that some genotypes (serotypes19A/ST320 and 18/ST1016) produced novel pneumococcal structures, predicting their roles in colony formation and adherence.

acute otitis media

community acquired pneumonia

choline‐binding protein A

clonal complex

Clinical and Laboratory Standards Institute

healthy children, prior to the administration of PCV7

healthy PCV7‐immunized children

multidrug resistance

minimum inhibitory concentration

multilocus sequence typing

pneumococcal adherent pili

pneumococcal conjugated vaccine

pulsed‐field gel electrophoresis

pneumococcal serine‐rich repeat protein

pneumococcal spike‐like structure

pneumococcal wire (pilus) structure

electron microscopy

sequence type

virulence‐related surface protein gene

Streptococcus pneumoniae, a leading bacterial human pathogen, has high morbidity and mortality rates 1. It colonizes the nasopharynx, particularly in children 1, 2, 3, nasopharyngeal colonization proceeding to development of pneumococcal diseases 1, 4, 5, 6. Invasive infections include bacteremia and meningitis; these are mostly reported in children aged <2 years 1. Mucosal/non‐invasive infections include non‐bacteremic pneumonia, AOM and sinusitis, which are less severe, but very common health issues 1, 6, 7. The most frequent S. pneumoniae infection is AOM, including patients in whom it is a complication of influenza 8.

The virulence factors of S. pneumoniae include capsular polysaccharides 9, 10, 11, 12, which represent the serotypes of S. pneumoniae; more than 90 serotypes have been identified 5, 11, 13. A polysaccharide capsule plays a key role in nasopharyngeal colonization and immune evasion from phagocytosis or complement factor binding 10, 12, 13, and is the target of PCVs such as PCV7 and PCV13 6, 11, 12, 13, 14.

Regarding pneumococcal surface proteins 9, 10, 11, 12, two types of pneumococcal pili, PI‐1 and PI‐2, have been identified. The major backbone subunit of PI‐1 is the RrgB protein, the expression of which (gene, rrgB) is regulated by the rlrA gene 15, 16, 17, 18. PI‐1 is present only in certain pneumococcal CCs. The backbone protein of PI‐2 is considered to be PitB (gene, pitB); the PI‐2 islet carries a second pilus gene pitA, which has a premature stop codon 19, 20. Although PI‐2 may be more widely distributed, it is weakly expressed 21.

The largest pneumococcal surface protein, PsrP, varies in size 22, 23. PsrP promotes pneumococcal adherence to nasopharyngeal epithelial cells and lung cells and contributes to robust formation of biofilms 9, 10, 22, 24, 25, 26. CbpA plays a role in adherence, colonization and immune evasion from complement attack and opsonophagocytosis 9, 10, 27.

S. pneumoniae forms biofilms during colonization of the nasopharynx and during diseases such as chronic otitis media and pneumonia 28, 29. Biofilms may endow pneumococci with reduced susceptibility to antimicrobial agents or resistance to immune recognition 10, 28. They may also contribute to colonization and persistence on the mucosa, but with altered virulence 24 or regulation 29.

S. pneumoniae serotypes/serogroups have been investigated in Russia 30, 31; three PCVs, PCV7, PCV10 and PCV13, have been registered for children. The molecular characteristics of S. pneumoniae have not yet been reported in Siberian Russia (Krasnoyarsk), which is located between the European and Far Eastern regions. We herein isolated and characterized S. pneumoniae in children before and after PCV7 vaccination and also in children with pneumococcal diseases in Krasnoyarsk. Because certain surface protein genes are highly variable, we designed PCR primers based on available pooled S. pneumoniae genome information. We also investigated pneumococcal surface ultrastructures using SEM.

MATERIALS AND METHODS

PCV7 vaccination, patients and ethics statement

None of the children in the present PCV7 trial had previously received S. pneumoniae vaccines. Furthermore, none of them received PCV7 before they presented with pneumococcal diseases. This study was approved by the Ethics Committee of Krasnoyarsk State Medical University (Protocol No. 2/2011). Informed consent was obtained from the parents or legal representatives of each child.

Typing of S. pneumoniae

Capsular typing was achieved by PCR, as previously described 32. STs were examined by PCR and sequencing, according to the MLST website 33 and CCs were analyzed using eBURST 34.

Susceptibility testing

The MICs of antimicrobial agents were measured by an agar dilution method using Mueller–Hinton agar (Difco, Sparks, MD, USA) supplemented with 5% sheep blood (Nippon Bio‐Test Laboratories, Tokyo, Japan), as described previously 35, 36. MICs of antimicrobial agents (including penicillin) for S. pneumoniae ATCC49619 (reference strain) that were obtained by the agar dilution method were consistent with those described by the CLSI 36. A disc method using antimicrobial agent discs (Becton Dickinson, Franklin Lakes, NJ, USA) was also employed (in initial susceptibility testing); oxacillin was used for penicillin in the disc method 36. Breakpoints for drug resistance were those described by CLSI 36. MDR was used when penicillin resistance (MIC ≥ 0.12 μg/mL for meningitis strains and MIC ≥ 8 μg/mL for non‐meningitis strains [intermediate, MIC 4 μg/mL]) and resistance (or intermediate) to at least two more antimicrobial agents were included.

Clonal analysis of S. pneumoniae

In PFGE analysis, bacterial DNA was digested with SmaI and electrophoresed in 1.2% agarose, as described previously 37.

Surface protein gene analysis by PCR

The four VSGs, PI‐1, PI‐2, psrP and cbpA, were analyzed by PCR. The primers used for PCR are summarized in Table S1 21, 38, 39 and are those reported by others plus those designed in the present study based on available pooled S. pneumoniae genome information (Fig. S1) 20, 21, 40, 41. The PCR conditions employed were initial denaturation at 95°C for 5 min, denaturation cycling at 95°C for 20 s, annealing at 52°C–55°C (depending on the Tm of primers used) for 30 s, extension at 68°C for 1 min (35 cycles), and a final extension at 68°C for 10 min, as described previously 39. PCR products were sequenced to confirm the target gene sequence.

Phylogenetic and homology analyses

A phylogenetic analysis was conducted in MEGA6 using the maximum likelihood method based on the Kimura 2‐parameter model; the reliability of the tree was estimated with a bootstrap analysis by 1000 replicates 42, 43. A homology analysis was performed using BLAST software 44 and DNAMAN 45.

Analysis of S. pneumoniae ultrastructures

To investigate bacterial surface structures, S. pneumoniae was grown on trypticase soy agar supplemented with 5% sheep blood (Becton Dickinson, Tokyo) at 37°C for 12–18 hr. Blood agar‐block pieces were fixed, dehydrated, critical‐point dried, coated with gold‐palladium and assessed using a SEM, as described previously 46. S. pneumoniae ATCC49619 was used as a control strain.

Adherence assay

Regarding adherence to HEp‐2 cells (a human epithelial cell line originating from human laryngeal carcinoma), S. pneumoniae cells, grown on blood agar plates as above, were added to HEp‐2 cells on plastic coverslips at 37°C for 2 hr, after which HEp‐2 cell samples were assessed by SEM, as described previously 47. S. pneumoniae ATCC49619 was used as a control strain.

Statistical analysis

Data were statistically analyzed using Fisher's exact test. The level of significance was defined as P < 0.05.

RESULTS

First PCV7 trial in Krasnoyarsk

One hundred and eighty‐three healthy children (aged 0–5 years; mean age, 3.7 years; 0.3% of the total population of approximately 70,000 children of corresponding ages in Krasnoyarsk), none of whom had any infectious diseases at the time of examination, were selected by pediatricians in 2011. Nasopharyngeal swabs were obtained from these healthy children prior to administration of PCV7 in 2011; these swabs yielded 93 S. pneumoniae strains. All 183 HC were then vaccinated with PCV7 in 2011. In 2013, nasopharyngeal specimens were obtained from 171 HIC; these swabs yielded 54 S. pneumoniae strains. The HIC did not receive antibiotics prior to the nasal swabs being obtained and S. pneumoniae isolated.

S. pneumoniae carriage rates in HC and HIC were 50.8% (93/183) and 31.6% (54/171), respectively, this being a significant decrease in carrier rate after vaccination (p < 0.01). S. pneumoniae carriage rates did not differ significantly between kindergarten‐attending and non‐attending children; most of the latter group of children had kindergarten‐ or school‐attending siblings. Although vaccinees included residents of orphanages, no significant bias in carriage rates was observed between these orphans and other children because the orphans were in various orphanages located in different parts of Krasnoyarsk and were thus not members of a closed group.

PCV7 serotypes in HC, such as 23F and 19F, were controlled by administration of PCV7; the number of serotypes/serogroups changed from eight (23F, 19F, 15AF, 23A, 14, 6, 35AC/42 and 18) to five (19A, 15BC, 6, 35AC/42 and 11AD) (Fig. 1), but with prominent emergence of the non‐PCV7 serotype 19A, confirming PCV7‐related serotype replacement over the two‐year period 48, 49, 50. There were no non‐encapsulated strains.

Figure 1

Serotype/serogroup distribution in the first small‐scale PCV7 trial in Krasnoyarsk, Siberian Russia. S. pneumoniae was isolated from the nasopharynxes of HC prior to administration of PCV7 in 2011 and then from HIC in 2013. Bars: blue, serotype/serogroup for HC; red, serotype/serogroup for HIC. Numbers above bars indicate percentages of each serotype/serogroup in the relevant group.

Because the entire study group in Krasnoyarsk was disbanded in 2012, this trial yielded no accurate data concerning vaccine efficacy regarding disease prevention. Regarding HC, the serotype coverage rates of PCV7 and PCV13 were 62.5%; in the present study, we examined serogroup 6 only (not 6A, 6B or 6C).

S. pneumoniae strains were also isolated from patients with pneumococcal diseases in three hospitals in Krasnoyarsk. The most prevalent pathogen of AOM, a common disease in children, was S. pneumoniae, which accounted for 32.6% of bacterial infections (23.8% of all cases) in Krasnoyarsk; this is consistent with previous findings 51, 52, 53, 54. The isolated strains included 12 strains from middle ear fluids of 12 patients with AOM (aged 8 months to 9 years; mean age, 2.0 years) in 2014 and 2016, four strains from aspirated sputum of four patients with CAP (aged 2 to 9 years; mean age, 6.2 years) in 2014–2016, and four strains from the blood of four patients with meningitis (aged 1 to 15 years; mean age, 8 years) in 2013–2015. Their serotypes/serogroups are summarized in Table 1. Regarding patients, the serotype coverage rates of PCV7 and PCV13 were 62.5% and 100%, respectively.

Table 1

Molecular characteristics of S. pneumoniae from children in Krasnoyarsk, Siberian Russia

						Virulence gene
Isolated from:	Isolation	Serotype or serogroup	ST (CC)	No. of strain	Resistance(resistant/total) i, intermediate	PI‐1	PI‐2	psrP	cbpA
Healthy children prior to PCV administration (n = 23)	2011	23F	1500 (30)	8		−	−	+	+
			30 (30)	1		−	−	+	+
			8636 † ( ‡ )	1		−	−	+	‐
		19F	236 (236)	1	P/Oi, E, C	+	−	−	+
			2323 (271)	1	P/Oi, Ei, T	+	+	−	+
			1203 (346)	1		−	−	−	+
		6	315 (315)	2	E,C,T	+	−	−	+
		35AC/42	1025 (1025)	2		−	−	+	+
		23A	8636† ( ‡ )	2		−	−	+	+
		14	9250 † ( ‡ )	2		−	−	−	+
		18	1016 (102)	1		−	−	−	+
		15AF	6202 (6202)	1		−	−	−	+
Children, immunized with PCV7 (n = 27)	2013	19A	320 (320)	11	P/Oi(10/11), E,C(10/11),T(10/11)	+	+	−	+
		6	315 (315)	3	E,C,T	+	−	−	+
			9248 † ( ‡ )	3		+	+	−	+
			9247 † ( ‡ )	1		+	+	−	+
	15BC		1025 (1025)	3		−	−	+	+
			9249 † ( ‡ )	2		−	−	+	+
	11 AD		62 (62)	2		−	−	−	−
	35AC/42		1025 (1025)	2		−	−	−	+
Patients
Acute otitis media	2014–2016	19A	320 (320)	5	P/O, E, C, Ti(1/5)	+	+	‐	+
(n = 12)		19F	1500 (30)	1		−	−	+	+
			271 (271)	1	P/O, E, C, T	+	+	−	+
			1464 (320)	2	P/O, E(1/2), C, T(1/2)	+	+	−	+
		23F	315 (315)	1	E,T	+	−	−	+
		9VA	156 (156)	1	P/O	+	+	−	+
		4	1637 (205)	1		+	−	−	+
Pneumonia	2014–2016	19F	2323 (271)	2	P/O, E,C	+	+	−	+
(n = 4)		19A	7915 (320)	2	P/O, E,C	+	+	−	+
Meningitis	2013–2015	3	505 (180)	1		−	−	−	+
(n = 4)		7AF	3544 (218)	1		−	+	−	+
		19F	9659 (320)	1	P/O, E,C,T	+	+	−	+
		6	5839 ( ‡ )	1		−	−	−	+

C, clindamycin; E, erythromycin; O, oxacillin; P, penicillin; T, tetracycline.

PI‐1+: rlrA + and rrgB +, rlrA +, or rrgB+; PI‐1−, rlrA − and rrgB −.

Novel ST found in Krasnoyarsk.

CC, not assigned.

Molecular characterization of S. pneumoniae

Twenty‐three HC and 27 HIC isolates were randomly selected from each serotype/serogroup and their molecular charcateristics examined; resultant data are summarized in Table 1. There were 11 ST types among the HC strains, the most prevalent being ST1500(CC30)/serotype23F, which was drug‐susceptible, whereas there were eight ST types among the HIC strains, the most prevalent being ST320(CC320)/serotype19A, which was MDR. MDR was observed only in CC320 and its related CC271 and CC236.

There were seven ST types among AOM strains (n = 27), the most prevalent being ST320/serotype19A, which was MDR (Table 1). CAP strains (n = 4) included CC271 and CC320; they were all MDR (Table 1). Meningitis strains (n = 4) were diverse in CCs; only CC320 was MDR (Table 1).

Regarding ST320/serotype19A strains, seven of the 10 HIC strains and two of the three AOM strains examined by PFGE (Fig. 2a) exhibited identical PFGE patterns (designated α1), two HIC strains showing only a one‐band difference (PFGE pattern α2), indicating the same clone (ST320/19AKras). Regarding CAP strains (Fig. 2b), two strains (ST7915/serotype19A) showed the PFGE α1 pattern, whereas the remaining two (ST2323/serotype19F) showed a two‐band difference (PFGE pattern α3) and belonged to the same clone (non‐invasive group) as ST320/19AKras. In contrast, meningitis strains exhibited divergent PFGE patterns (Fig. 2b).

Figure 2

PFGE analysis of (a) Comparison of ST320/serotype19A S. pneumoniae from patients with AOM and healthy HIC. (b) Comparison of S. pneumoniae from patients with pneumonia and meningitis with representative PFGE types of ST320/serotype19A S. pneumoniae. Color in strain column: pink, AOM isolates; green, HIC isolates; orange, pneumonia isolates; blue, meningitis isolates. M, molecular size standard (lambda ladder).

Regarding VSGs (Tables 1 and S2), when the 70 strains (Table 1) were examined, PI‐1, PI‐2, psrP and cbpA were found to be present at 54.3% (38/70), 38.6% (27/70), 48.6% (34/70), and 95.7% (67/70), respectively (Table 2). There were two major SVG content types, PI‐1−/PI‐2−/psrP +/cbpA + and PI‐1+/PI‐2+/psr −/cbpA +; the latter correlated with a non‐invasive AOM and CAP group (Table 2), mainly in association with CC320 and CC271 (Fig. 3). In contrast, the PI‐1−/PI‐2−/psrP +/cbpA + type correlated with the colonization of HC (Table 2), mainly in association with CC30 and CC1025 (Fig. 3).

Table 2

Distribution of virulence‐related surface protein genes (VSGs) and VSG content types in S. pneumoniae isolates

S. pneumoniae‐isolation group (number of children)	PCR‐positive percent of the gene/region (positive/total)
	PI‐1	PI‐2	psrP	cbpA
Healthy children (HC), prior	17% (4/23)	4% (1/23)	87% (20/23)	96% (22/23)
to the PCV7 administration
(n = 23)
Healthy PCV7‐immunized	67% (18/27)	41% (11/27)	44% (12/27)	93% (25/27)
children (HIC) (n = 27)
Disease, total (n = 20)	80% (16/20)*1	75% (15/20)*1	10% (2/20)*1	100% (20/20)
acute otitis media (n = 12)	92% (11/12)*2	75% (9/12)*2	17% (2/12)*2	100% (12/12)
Pneumonia (n = 4)	4/4	4/4	0/4	4/4
acute otitis media+	94% (15/16)*3	81% (13/16)*3	13% (2/16)*3	100% (16/16)
pneumonia (n = 16)
Meningitis (n = 4)	1/4	2/4	0/4	4/4
P value, vs. that of HC	*1, P < 0.01	*1, P < 0.01	*1, P < 0.01
	*2, P < 0.01	*2, P < 0.01	*2, P < 0.01
	*3, P < 0.01	*3, P < 0.01	*3, P < 0.01

Figure 3

Distribution of virulence‐related surface protein genes (regions), PI‐1, PI‐2, psrP and cbpA, for each (a) PI‐1; (b) PI‐2; (c) psrP; (d) cbpA. Histograms show the numbers of gene‐positive/total strains, stratified by the CC or ST; positive‐strain numbers are shown by dark‐colored boxes. CCs were not assigned for the ST types shown.

Ultrastructures of S. pneumoniae

S. pneumoniae cells grown on blood agar were analyzed by SEM (Fig. 4). When ST320/19AKras‐α1 (Fig. 2a) was examined, very long PWS were found in the colonies (Fig. 4a, b). PWS were > 4 μm in length, gently curved, and had the appearance of whips or even peritrichous flagella; no short pili were observed. In the case of ST320/19AKras‐α2 (Fig. 2a), S. pneumoniae cells in colonies only had shorter, straight, thin pili (Fig. 4c). S. pneumoniae ATCC49619, used as a control strain, showed no PWS or obvious pili.

Figure 4

Scanning electron micrographs showing cell surface structures of ST320/serotype19A (a, b) PFGE α1 type (ST320/19AKras‐α1, Fig. 2a) of ST320/serotype19A was examined; S. pneumoniae has gently curved PWS (arrow). (c) PFGE α2 type (ST320/19AKras‐α2, Fig. 2a) was examined; S. pneumoniae have short, thin, straight pili (arrow).

When S. pneumoniae in HC (Table 1) was examined in the HEp‐2 cell assay (Fig. 5), each single cell of ST1016/serogroup18 (PI‐1−/PI‐2−) was clearly piliated (Fig. 5a, arrow) and showed pili‐mediated adherence (Fig. 5c); these pili (PAP) were often observed to form a ring at the position of cell septation sites (Fig. 5d; arrow). In contrast, no obvious pili‐mediated adherence to HEp‐2 cells was found for other S. pneumoniae in HC. S. pneumoniae belonging to ST320/19A Kras‐α1 and α2 (Fig. 2a) and S. pneumoniae ATCC49619, used as a control strain, also showed no adherence to HEp‐2 cells (data not shown).

Figure 5

Scanning electron micrographs showing cell surface structures of ST1016/serogroup18 (a) Single ST1016/serogroup18 cell with pili (arrow) on a plastic coverslip. (b) HEp‐2 cell. (c, d) Adherence of ST1016/serogroup18 cells to HEp‐2 cells with PAP (arrow).

DISCUSSION

In Krasnoyarsk, Siberian Russia, S. pneumoniae found to be colonizing healthy children (HC and HIC) included five novel ST types (ST8636, ST9247, ST9248, ST9249 and ST9250), representing Krasnoyarsk (Siberia)‐local characteristics. S. pneumoniae in HC, examined in 2011, was mostly drug‐susceptible, the most prevalent type being ST1500(CC30)/serotype23F with the SVG content type PI‐1−/PI‐2−/psrP +/cbpA +. The genotype psrP + in HC is consistent with previous reports that PsrP promotes nasopharyngeal pneumococcal colonization 25, 55. Although the serotypes were divergent, ST1500 was also a minor constituent of isolates from patients with AOM (between 2014 and 2016). CC30 is not a global CC and, to the best of our knowledge, there is currently no information available on its genome. A few of the S. pneumoniae colonizing HC were MDR; ST236(CC236)/serotype19F and ST2323(CC271)/serotype19F were found to be related to MDR ST320(CC320)/serotype19A (Figs. S2, S3).

The first small‐scale PCV7 trial in Krasnoyarsk resulted in serotype replacement with global, MDR ST320/serotype19A (Fig. S3), which is consistent with previous findings 48, 49, 50, 56, 57, 58. Nasopharyngeal colonization precedes any type of pneumococcal disease, including AOM. PCV7 primarily increases serotype 19A carriage and, as a consequence, increases the rate of 19A‐related pneumococcal disease. In the present study, because HIC did not receive antibiotics prior to isolation of S. pneumoniae, the underlying mechanism or source of emergence in HIC of ST320/serotype19A, which appeared to have a high fitness cost from MDR 59, currently remains unknown. ST320/serotype19A may have superior fitness to the “empty” nasopharyngeal niche in HIC. Moreover, in the present study we did not examine carriage in healthy children who had not been vaccinated with PCV7.

Of the four VSGs, PI‐1, PI‐2, psrP and cbpA are located at a region (named rlrA pathogenicity islet or PI‐1 islet) flanked by IS1167 21, 60, the PI‐2 islet 20, 21, a pathogenicity island 22, 26, 61, and a chromosomal core (in some cases, a region flanked by IS) 41, 62, respectively. Certain VSGs had highly variable sequences, confirming previous findings 41. Therefore, we improved the PCR primers and also attempted to use multiple primers for each PCR target. As a result, the low yields (for example, 55.7%‐positive) in PCR using previously reported primers increased to high yields (for example, 94.3%) in the present study, reaching the level predicted on the basis of pooled genome sequences previously reported. However, a limitation of the present study was that we only analyzed four VSG targets and the disease groups studied, particularly CAP and meningitis, were small. Results of PFGE analysis and VSG content typing strongly suggest that AOM and CAP isolates are a common non‐invasive group. The results of the present study also indicate that the surface proteins PitA and PitB are protected from attacks by host immunity because, even though rrgB, psrP and cbpA were highly variable, pitA and pitB were conserved.

In previous studies, we found that it was possible to demonstrate the unique surface structures of bacterial pathogens in colonies (at high cell densities) using SEM 46, 63, 64. In the present study, we found that the major epidemiological clone (PFGE α1 type) of ST320/serotype19A, associated with AOM, formed novel PWS in colonies; the PWS were unusually long, curved, and did not have the morphological appearance of pili, instead resembling whips or peritrichial flagella. AOM‐associated ST320/serotype19A was PI‐1+/PI‐2+. The molecular and genetic features of PWS are currently being investigated. Type IV pili, very long pilus structures, have been investigated in gram‐negative bacteria 65. We also noted heavy biofilm formation and PSS in colonies of AOM‐associated ST320/serotype19A (Figs. S4, S5). At high cell densities in colonies, these structures may adhere together to make rigid, non‐invasive clinical foci on the mucosa, thus facilitating successful colonization and infection.

We also found that, in HC, ST1016/serogroup18 formed HEp‐2 cell‐adherent pili. Given that ST1016/serogroup18 was PI‐1−/PI‐2−, PAP represent the third most frequent adherent pili in S. pneumoniae that play roles in colonization of HC.

In conclusion, we, for the first time, here report changes in pneumococcal serotype distribution in a cohort of children after PCV7 vaccination and in a small group of patients with pneumococcal disease in Krasnoyarsk, Siberian Russia. We found Krasnoyarsk (Siberia)‐local clones. The most prevalent S. pneumoniae in the nasopharyngeal niche of HC was drug‐susceptible ST1500/serotype23F with the VSG content type PI‐1−/PI‐2−/psrP +/cbpA +, suggesting that PsrP plays an important role in nasopharyngeal colonization. PCV7 increased the carriage in HIC of MDR serotype 19A/ST320, with PI‐1+/PI‐2+/psrP ‐/cbpA + and, as a consequence, increased the rate of 19A/ST320‐related non‐invasive pneumococcal disease, including AOM, in non‐vaccinated children. The major epidemiological clone of ST320/serotype19A (PI‐1+/PI‐2+) formed novel PWS in colonies, most likely for rigid colonization and immune evasion in the middle ear. ST1016/serogroup18 (PI‐1−/PI‐2−) in HC formed novel HEp‐2 cell‐adherent pili, possibly for nasopharyngeal colonization and was the third most frequent pneumococcal pili.

DISCLOSURE

The authors declare that they have no conflicts of interest.

Additional supporting information may be found in the online version of this article at the publisher's web‐site.

Supporting Figure S1‐1.

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Supporting Figure S1‐2.

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Supporting Figure S1‐3.

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Fig S1‐4. Structures of virulence‐related surface protein genes, cbpA, rrgB, pitA, pitB and psrP, and the new PCR primer design. (a) cbpA. (b) rrgB. (c1) pitA. (c2) pitB. (d1 to d3) psrP. () Phylogenetic tree analysis. () Nucleotide sequence comparison. () Amino acid sequence comparison. () Strain types are shown as ST (CC)/serotype. Strains marked in yellow were analyzed for conserved or divergent regions at the nucleotide sequence () and amino acid sequence () levels. In () and (), homologous regions are shaded. Abbreviations in (): Cna_B, Cna protein B‐type domain; DUF11, domain of unknown function (gram‐positive pilin backbone subunit 2); GramPos_pilinBB (DUF11 super family); GramPos_pilinD1, gram‐positive pilin subunit D1; GramPos_pilinD3, gram‐positive pilin backbone subunit 3; LPXTG, LPXTG motif for cell wall anchoring (in b‐, d1‐, d2‐, d3‐); RICH, Rich In CHarged residues (in a‐); VTPTG, the VTPTG motif (in c2‐); VWA_2, von Willebrand factor type A domain (in c1‐). New PCR primers were designed to target the conserved region of each gene. The PCR primers indicated in () are described in Table S1. Regarding cbpA (a), the cbpA sequence varied greatly. Although the primer cbpA‐F/cbpA‐R was designed within a conserved region, when the 70 strains (Table 1) were examined, the primer JVS73L/JVS74R sequences targeted a less homologous region (a‐, ), yielding 94.3%‐positive and 55.7%‐positive results, respectively. Regarding rrgB (b), rrgB encodes the major backbone subunit, RrgB, of PI‐1. rrgB exhibits a limited number of clusters in a phylogenetic analysis (b‐), but with a highly divergent sequence for each cluster (b‐, ). Although the primer rrgB‐F/rrgB‐R was designed within a conserved region, the primer JVS69L/JVS70R targeted a highly variable region, yielding 52.9%‐positive and 40.0%‐positive results, respectively, for the 70 strains (Table 1). Regarding pitA (c1) and pitB (c2), pitA and pitB are located in PI‐2 islet; the pitB product, PitB, is the backbone protein of PitB pilus. A second pilus gene pitA has a premature stop codon 20, 21. pitA of ST320/19A strains from AOM in the present study also had a premature stop codon. pitA and pitB also exhibit a limited number of clusters in the phylogenetic analysis (c1‐, , c2‐, ). However, each gene sequence is highly conserved (c1‐; c2‐). The pitA primers, P06 for/P06 rev, pitA‐F/pitA‐R, and pitB‐F/pitB‐R, all yielded 38.6% for the 70 strains (Table 1). Regarding psrP (d1) to (d3), psrP sequences were classified into two major groups, about 14 kpb psrP (d1) and about 4 kb psrP (d2), designated as psrP(S). In (d3), psrP and psrP(S) were compared, revealing a marked difference in the size of the serine‐rich repeat region on the C‐terminal side. psrP (about 14 kpb in size) is highly variable according to phylogenetic analysis (d1‐). The primer JVS77L/JVS78R targets the conserved region on the 3′‐terminal (C‐terminal) side, whereas the primer psrP‐F/psrP‐R targets that on the 5′‐terminal (N‐terminal) side (d1‐, ). The target sequence of the primer JVS77L/JVS78R is present in 8/10 genome/psrP sequences searched and absent in 2/5 genome/psrP(S) sequences. However, that of the primer psrP‐F/psrP‐R is present in all sequences searched, including psrP(S). The primers psrP‐F/psrP‐R and JVS77L/JVS78R yielded 48.6% and 28.6%‐positive results, respectively, for the 70 strains (Table 1). All CC320 (and its related CC236 and CC271) strains in the present study were negative for psrP in PCR, which is consistent with the negative results obtained in our search of reported CC320 genome sequences; similar negative findings have previously been reported 40, 41.

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Fig S2. Genetic relationships of CC236/CC271/CC320 lineages of S. pneumoniae in Krasnoyarsk, Siberian Russia. Of the 70 strains (Table 1) examined, 26 (37.1%, 26/70) belonged to the complex CC271/CC236/CC320 lineages, including ST types (236, 271, 320, 1464, 2323, 7915 and 9659). (a) ST types and their allelic profiles of the CC271/CC236/CC320 strains of S. pneumoniae are shown. Among the seven genes (aroE, gdh, gki, recP, spi, xpt and ddl) used for ST typing (allelic profiling), ddl varied the most, resulting in four types: ddl1, ddl26, ddl106, and ddl610. aroE and xpt are occasionally responsible for additional variations. (b) The nucleotide sequences of the ddl genes were analyzed for phylogenetic diversity. (c) The genetic relationship of each ST type is shown for serotype 19F or serotype 19A.

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Fig S3. Isolation and distribution of CC236/CC271/CC320 lineages of S. pneumoniae in Krasnoyarsk, Siberian Russia. The CC236/CC271/CC320 lineages, including ST types (236, 271, 320, 1464, 2323, 7915 and 9659) that were analyzed as shown in Fig. S2, were isolated in a PCV7 trial before and after PCV7 (a, b) and from non‐immunized pediatric patients with pneumococcal diseases (a, c). MDR ST320/serotype19A (marked in green) was isolated from HIC in 2013 and from patients with AOM in 2014–2016. MDR ST232/serotype19F (marked in orange) was isolated from HC prior to administration of PCV7 in 2011 and from patients with pneumonia in 2014–2016.

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Fig S4. Scanning electron micrographs showing a colony of ST320/serotype19A S. pneumoniae on blood agar plates. The PFGE α1 type (ST320/19AKras‐α1, Fig. 2a) was examined. The central region of the colony (a) had very heavy biofilms (b and c). (c) Arrow, S. pneumoniae in heavy biofilms. S. pneumoniae at the colony center (near the edge) has PWS. (b) On the left, an arrow indicates S. pneumoniae with PWS. In contrast, S. pneumoniae cells located outside of colonies (near the edge) had no detectable PWS (right arrow in b).

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Fig S5. Scanning electron electronmicrograph showing PSS. S. pneumoniae, serotype19A/ST320 PFGE α1 type (19A/ST320Kras‐α1, Fig. 2a), was grown on blood agar. Arrow, PSS; arrowhead, PWS.

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Table S1. Primers used for the PCR assay

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Table S2. Yields in PCR for each primer

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