Highlights
- •A total of 67.6% of nonencapsulated S. pneumoniae (NESp) were nonsusceptible to penicillin.
- •Penicillin-binding proteins (PBPs) of 71 NESp isolates were genetically analyzed.
- •To the best of our knowledge, all the PBP profiles (1a-2b-2x) that were identified were novel.
- •NESp had more diverse mutations in PBPs than encapsulated S. pneumoniae.
- •Various mutations in PBPs may be related to penicillin nonsusceptibility in NESp.
Abstract
Objectives
Nonencapsulated Streptococcus pneumoniae (NESp) is emerging after the introduction of pneumococcal conjugate vaccines (PCVs). This study aimed to elucidate the genetic characteristics of penicillin-binding proteins (PBPs; PBP1a, 2b, and 2x) associated with penicillin nonsusceptibility in emergent NESp.
Methods
A total of 71 NESp isolates that were identified in our previous study during the PCV era in Japan (2011–2019) were analyzed for their amino acid sequences of transpeptidase domain in PBP 1a, 2b, and 2x.
Results
Overall, we identified 21 different PBP profiles (1a-2b-2x), all of which represent novel PBP profiles. The dominant PBP profiles were 13-16-ne1 (32.4%, n = 23), ne1-16-ne2 (14.1%, n = 10), and 13-7-ne4 (7.0%, n = 5) (novel PBP type was numbered with “ne” denoting “nonencapsulated”), accounting for 53.5% of all isolates. All isolates with the PBP profiles 13-16-ne1 and 13-7-ne4 and those having PBP1a type-13 and -131, PBP2b type-7, -ne1, and -ne2 showed nonsusceptibility to penicillin. A high degree of genetic diversity was found in PBP2x, with most of them (81.7%) being new types.
Conclusions
Our current study identified the 21 novel PBP profiles and remarkable mutations in the PBPs, which may be potentially associated with penicillin nonsusceptibility in NESp.
Keywords
Abbreviations:
NESp (nonencapsulated Streptococcus pneumoniae), PCVs (pneumococcal conjugate vaccines), PBPs (penicillin-binding proteins), TP (transpeptidase), aa (amino acid), ST (sequence type), MICs (minimum inhibitory concentrations), ESp (encapsulated S. pneumoniae), PEN (penicillin), S (susceptible), I (intermediate), R (resistance), A (Alanine), N (Asparagine), D (Aspartate), E (Glutamate), Q (Glutamine), G (Glycine), H (Histidine), I (Isoleucine), L (Leucine), K (Lysine), M (Methionine), F (Phenylalanine), P (Proline), S (Serine), T (Threonine), Y (Tyrosine), V (Valine)1. Introduction
After the widespread use of pneumococcal conjugate vaccines (PCVs) in children, nonencapsulated S. pneumoniae (NESp) has emerged among the pneumococci (
Valentino et al., 2014
). We recently reported on the clonal lineages and antimicrobial resistance of NESp in Japan and noted the high prevalence rates of nonsusceptibility to penicillin (67.6%) (Kawaguchiya et al., 2021
). Resistance to β-lactams is mainly caused by alterations in the transpeptidase (TP) domain of the penicillin-binding proteins (PBPs; PBP1a, 2b, and 2x), encoded by the pbp1a, pbp2b, and pbp2x genes. These PBPs contain conserved amino acid (aa) motifs (SXXK, SXN, and KXG), which are responsible for TP activity (Granger et al., 2005
; Granger et al., 2006
). In this study, we analyzed the genetic diversity in PBPs of penicillin-susceptible and penicillin-nonsusceptible NESp in the post-PCV era in Japan.2. Methods
In our previous study, we reported that during the PCV era (from the PCV7–PCV13) in Japan (2011–2019), 71 NESp isolates were identified from a total of 4463 pneumococcal clinical isolates, and their sequence type (ST) were determined (
Kawaguchiya et al., 2021
). Among the NESp isolates, 48 isolates (67.6%) showed penicillin nonsusceptibility (intermediate-resistance (33.8%, n = 24) and resistance (33.8%, n = 24), showing MIC of 0.12–1.0 and ≥ 2 µg/mL, respectively). In the current study, we examined the TP domain aa sequences of PBP1a, 2b, and 2x in all 71 NESp isolates through the DNA sequencing of the PCR-amplified products as described previously (Granger et al., 2005
; Granger et al., 2006
). The conserved motifs in the TP domain regions for PBP1a (370STMK373, 428SRNVF432, 557KTG559, 574TSQF577), PBP2b (385SVVK338, 442SSNT445, 614KTGTA618), and PBP2x (337STMK340, 394HSSN397, and 546LKSGT550) were analyzed (Granger et al., 2005
; Granger et al., 2006
). PBP types were identified using the PBP type sequence database of the CDC Streptococcus Laboratory (http://www.cdc.gov/streplab/mic-tables.htm.). Genetic analysis was performed for the TP domain sequences of the 3 pbp genes along with that of penicillin-susceptible S. pneumoniae R6 strain (GenBank accession no. NC_003098.1). The PBP TP domain sequences in this study were deposited in the GenBank database under accession numbers OK166972 to OK167002 (Supplementary Tables 1–3).3. Results and discussion
In this study, we identified 3 novel PBP types (numbered with “ne”, denoting “nonencapsulated”) for PBP1a and 2b and 8 novel PBP types for 2x (Table 1). The genetic analysis revealed the presence of 21 different PBP profiles (1a-2b-2x), all of which represent new profiles, with 13-16-ne1 (32.4%, n = 23), ne1-16-ne2 (14.1%, n = 10), and 13-7-ne4 (7.0%, n = 5) being the most prevalent. All isolates with PBP type 13-16-ne1 were nonsusceptible to penicillin. In addition, most of the isolates (82.6%, n = 19/23) belonged to ST4845 or ST11379, which represent a single or double locus variant, respectively of the Denmark14-ST230 global clone. Because none of the 21 PBP profiles have been detected in encapsulated clones previously (
Li et al., 2016
; Chen et al., 2020
), the PBP variants were suggested to have emerged from NESp lineages.Table 1PBP profile, MICs to antimicrobials, and STs of NESp isolates.
| PBP profile | No. (%) of isolates | No. (%) of isolates with MIC to penicillin (μg/mL) | ST (MLST)(no. of isolates) | ||||
|---|---|---|---|---|---|---|---|
| 1a-2b-2x | ≤ 0.06 | 0.12 | 0.5 | 1.0 | 2.0 | ||
| 13-16-ne1 | 23 (32.4) | 0 | 0 | 0 | 4 (17.4) | 19 (82.6) | 4845 (12), 11379 (7), 7502 (2), 16217 (1), 16221 (1) |
| ne1-16-ne2 | 10 (14.1) | 6 (60.0) | 2 (20.0) | 2 (20.0) | 0 | 0 | 7502 (10) |
| 13-7-ne4 | 5 (7.0) | 0 | 1 (20.0) | 0 | 3 (60.0) | 1 (20.0) | 16214 (5) |
| 1-4-ne2 | 4 (5.6) | 4 (100) | 0 | 0 | 0 | 0 | 7786 (4) |
| 2-4-ne3 | 3 (4.2) | 3 (100) | 0 | 0 | 0 | 0 | 3288 (3) |
| 86-16-126 | 3 (4.2) | 2 (66.7) | 0 | 0 | 0 | 1 (33.3) | 7494 (3) |
| 131-ne1-154 | 3 (4.2) | 0 | 0 | 0 | 2 (66.7) | 1 (33.3) | 9043 (1), 16220 (2) |
| ne1-16-126 | 3 (4.2) | 3 (100) | 0 | 0 | 0 | 0 | 7502 (3) |
| ne2-4-ne3 | 3 (4.2) | 3 (100) | 0 | 0 | 0 | 0 | 16217 (3) |
| 1-7-497 | 2 (2.8) | 0 | 2 (100) | 0 | 0 | 0 | 16214 (2) |
| 181-ne2-36 | 2 (2.8) | 0 | 0 | 2 (100) | 0 | 0 | 7803 (1), 8463 (1) |
| 1-16-8 | 1 (1.4) | 0 | 1 (100) | 0 | 0 | 0 | 2809 (1) |
| 1-0-ne2 | 1 (1.4) | 1 (100) | 0 | 0 | 0 | 0 | 7786 (1) |
| 13-16-ne8 | 1 (1.4) | 0 | 0 | 0 | 1 (100) | 0 | 16223 (1) |
| 13-ne3-ne1 | 1 (1.4) | 0 | 0 | 0 | 1 (100) | 0 | 4845 (1) |
| 146-48-ne6 | 1 (1.4) | 0 | 1 (100) | 0 | 0 | 0 | 16214 (1) |
| ne1-16-55 | 1 (1.4) | 0 | 0 | 1 (100) | 0 | 0 | 4845 (1) |
| ne1-16-ne1 | 1 (1.4) | 0 | 0 | 0 | 0 | 1 (100) | 16216 (1) |
| ne1-16-ne5 | 1 (1.4) | 0 | 0 | 1 (100) | 0 | 0 | 7502 (1) |
| ne1-16-ne7 | 1 (1.4) | 0 | 0 | 0 | 0 | 1 (100) | 4845 (1) |
| ne3-16-ne2 | 1 (1.4) | 1 (100) | 0 | 0 | 0 | 0 | 7502 (1) |
| Total | 71 (100) | 23 (32.4) | 7 (9.9) | 6 (8.4) | 11 (15.5) | 24 (33.8) | |
NESp, nonencapsulated Streptococcus pneumoniae; PBP, penicillin binding protein; MIC, minimum inhibitory concentration; ST, sequence type; MLST, multilocus sequence typing.
a Identified as a new PBP type in the present study, shown by numbered with “ne” denoting “nonencapsulated”.
b Susceptible, intermediate, and resistant MIC breakpoints for penicillin were ≤ 0.06, 0.12-1, and ≥ 2 µg/mL, respectively using the Clinical and Laboratory Standards Institute MIC definitions for oral penicillin V.
Genetic analysis of PBP1a revealed the presence of 10 genetic types, among which type-13 was the most common (42.3%, n = 30), followed by type-ne1 (23.9%, n = 17) (Supplementary Table 1). All isolates with PBP1a type-13, type-131, type-146, and type-181 were nonsusceptible to penicillin, whereas those with type-ne1 showed a lower nonsusceptibility rate to penicillin (47.1%, n = 8/17). Similarly, high resistance rates to β-lactams have been reported for invasive pneumococcal strains with PBP1a type-13 (
Li et al., 2016
; Metcalf et al., 2016
; Chen et al., 2020
; Varghese et al., 2021a
). Between PBP1a type-13 and type-ne1, there are only 4 aa differences at positions 533, 540, 546, and 550, located near the 557KTG559 motif, and these 4 aa substitutions were also detected in type-131. Moreover, isolates with PBP 1a type-146 and type-181, with 3 of 4 substitutions, were also nonsusceptible to penicillin, similar to the IPD penicillin-nonsusceptible pneumococcal strain (Zhou et al., 2016
). In agreement with a previous report (Varghese et al., 2021b
), the P432T mutation in the 428SRNVP432 motif was also found together with the T371A mutation in the 370STMK373 and TSQF574–577NTGY substitutions in PBP1a type-13, -131, -ne1, and -ne3 isolates (Supplementary Table 1). Accordingly, accumulation of these aa substitutions was suggested to be involved in penicillin nonsusceptibility in isolates with PBP1a type-13 and type-131.Among PBP2b, type-16 (64.8%, n = 46) was the most prevalent, followed by type-4 (n =10) and type-7 (n = 7) (Supplementary Table 2). All isolates with PBP2b type-7 belonging to ST16214, as well as those with type-ne1 and type-ne2, showed nonsusceptibility to penicillin. Only type-7 had 7 unique substitutions (A250S, D624G, Q627E, T629N, Q673N, K674Q, and Y675H), which were implicated in high-level penicillin resistance (
Zhou et al., 2016
). Regarding PBP2x, a high prevalence of new types (ne1–ne8) (81.7%) was noted (Supplementary Table 3), with type-ne1 and type-ne4 being associated with penicillin nonsusceptibility. Because pbp2x is located close to the cps (capsule polysaccharide synthesis) locus, greater diversity of pbp2x may be related to deletion event of cps in NESp, as described for recombination in cps, causing serotype switching (Dewé et al., 2019
).In conclusion, all of the NESp isolates showed novel PBP profiles, including unique PBP1a, 2b, and 2x types, among which specific types were more likely to be involved in penicillin resistance. Because NESp was confirmed to have more divergent PBPs than encapsulated S. pneumoniae, continued genetic surveillance of NESp is necessary in the postvaccine era.
Ethical statement
In this study, no human participants were involved directly. Hence, human ethics clearance was not required. We used pneumococcal isolates routinely cultured from clinical specimens in hospitals and clinics.
Funding
This research was supported by Japan Society for the Promotion of Science KAKENHI, Grant No. 19K10603.
Declaration of Competing Interest
The authors declare no competing interests.
Appendix A. Supplementary materials
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Article info
Publication history
Published online: April 22, 2022
Accepted:
April 14,
2022
Received in revised form:
March 14,
2022
Received:
February 9,
2022
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