Genomic analysis of penicillin-binding proteins and recombination events in an emerging amoxicillin- and meropenem-resistant PMEN3 (Spain9V-3, ST156) variant in Taiwan and comparison with global descendants of this lineage

ABSTRACT From 2008 to 2020, the Taiwan National Notifiable Disease Surveillance System database demonstrated that the incidence of non-vaccine serotype 23A invasive pneumococcal disease (IPD) approximately doubled. In this study, 276 non-repetitive pneumococcal clinical isolates were collected from two medical centers in Taiwan between 2019 and 2021. Of these 267 pneumococci, 60 were serotype 23A. Among them, 50 (83%) of serotype 23A isolates belonged to the sequence type (ST) 166 variant of the Spain9V-3 clone. Pneumococcal 23A-ST166 isolates were collected to assess their evolutionary relationships using whole-genome sequencing. All 23A-ST166 isolates were resistant to amoxicillin and meropenem, and 96% harbored a novel combination of penicillin-binding proteins (PBPs) (1a:2b:2x):15:11:299, the newly identified PBP2x-299 in Taiwan. Transformation of the pbp1a, pbp2b, and pbp2x alleles into the β-lactam-susceptible R6 strain revealed that PBP2x-299 and PBP2b-11 increased the MIC of ceftriaxone and meropenem by 16-fold, respectively. Prediction analysis of recombination sites in PMEN3 descendants (23A-ST166 in Taiwan, 35B-ST156 in the United States, and 11A-ST838/ST6521 in Europe) showed that adaptive evolution involved repeated, selectively favored convergent recombination in the capsular polysaccharide synthesis region, PBPs, murM, and folP genome sites. In the late 13-valent pneumococcal conjugate vaccine era, PMEN3 continuously displayed an evolutionary capacity for global dissemination and persistence, increasing IPD incidence, leading to an offset in the decrease of pneumococcal conjugate vaccine serotype-related diseases, and contributing to high antibiotic resistance. A clonal shift with a highly β-lactam-resistant non-vaccine serotype 23A, from ST338 to ST166, increased in Taiwan. ST166 is a single-locus variant of the Spain9V-3 clone, which is also called the PMEN3 lineage. All 23A-ST166 isolates, in this study, were resistant to amoxicillin and meropenem, and 96% harbored a novel combination of penicillin-binding proteins (PBPs) (1a:2b:2x):15:11:299. PBP2x-299 and PBP2b-11 contributed to the increasing MIC of ceftriaxone and meropenem, respectively. Prediction analysis of recombination sites in PMEN3 descendants showed that adaptive evolution involved repeated, selectively favored convergent recombination in the capsular polysaccharide synthesis region, PBPs, murM, and folP genome sites. In the late 13-valent pneumococcal conjugate vaccine era, PMEN3 continuously displays the evolutionary capacity for dissemination, leading to an offset in the decrease of pneumococcal conjugate vaccine serotype-related diseases and contributing to high antibiotic resistance.


Whole-genome sequencing and assembly
To study the evolutionary relationships of 23A-ST166 in Taiwan, we used 52 S. pneu moniae clinical isolates to perform whole-genome sequencing (WGS), including 50 23A-ST166 isolates collected during 2019-2021, and one each of 9V-ST156 and 9V-ST166 identified in our previous study (19,20).Genomic DNA of pneumococci was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany), and sequencing libraries were prepared for WGS using NEBNext Ultra II DNA Library Prep Kits (Illumina, USA) with Illumina MiSeq for 600 cycles (2 × 150 bp paired-end).The raw read quality assessment was performed using FastQC version 0.11.3.The contig was assembled using SPAdes version 3.13.0(21) and evaluated using QUAST (22) with an N 50 length of 87,487 bp.All WGS data in this study were deposited in the Sequence Read Archive (SRA) database under the BioProject PRJNA852704 (Table S1).

Comparing genomic variation of PMEN3 descendants from different countries
The genomic characteristics of 23A-ST166 isolates from Taiwan were compared with those of isolates from European countries and the United States.WGS data were downloaded from the NCBI SRA database (BioProject: PRJEB24965, PRJNA284954, and PRJEB32798).Draft genome data, from 14 35B-ST156 and 49 11A-ST838/ST6521 isolates, were collected for subsequent analysis (15,16).

Predicting recombination sites by phylogenetic analysis
WGS trimmed reads were aligned against the ATCC-700671 (9V-ST156, GenBank accession no: CP099641) as described previously (2), and the resulting phylogenetic tree, isolate metadata, core genome SNPs, and recombination sites were visualized by using Phandango version 1.3.0(23).

Penicillin-binding protein profiles
To compare the sequences of the transpeptidase regions of the pbp1a, pbp2b, and pbp2x genes of 23A-ST166, in silico PBP profiles were determined using BLASTX by aligning the assembly nucleotide contig with the U.S. CDC PBP database (https://www.cdc.gov/streplab/pneumococcus/mic.html).

Penicillin-binding protein transformation
The non-encapsulated laboratory strain R6, an amoxicillin-susceptible pneumococcus (MIC 0.016 mg/L), was used as the recipient in PBP transformation studies.The entire pbp1a, pbp2b, and pbp2x genes of the 23A-ST166 clinical isolates were PCR amplified and then cloned into pJET1.2/blunt(Thermo Fisher, USA) according to the manufacturer's instructions (2).The pbp-containing plasmid was transformed into the S. pneumoniae R6 strain using CSP-1 (2) and then spread on Mueller-Hinton agar containing 5% sheep blood and different concentrations of amoxicillin.After 24 h of incubation, colonies were selected from plates containing the highest amoxicillin concentration.The pbp gene of the transformants was PCR amplified and Sanger sequenced.The MICs of antibiotics in the selected transformants were analyzed according to CLSI guidelines.

Statistical analysis
SPSS 15.0 for Windows (Statistical Package for Social Sciences, Chicago, IL, USA) was used for statistical analysis.Statistical comparisons of the incidence were performed using Poisson distribution with 95% confidence intervals.Differences were statistically significant at P < 0.05.

Serotype replacement with the increase of serotype 23A-associated IPD from 2010 to 2020
Surveillance data from the TCDC IPD surveillance system showed that the total incidence of IPD significantly decreased from 3.5 episodes/100,000 persons/year in 2008 to 0.97 episodes/100,000 persons/year in 2020 after the PCV13 vaccination program (P < 0.001).However, the incidence of IPD caused by serotype 23A increased from 0.15 episodes/100,000 persons/year in 2008 to 0.25 episodes/100,000 persons/year in 2019 (P = 0.011) and then decreased to 0.13 episodes/100,000 persons/year in 2020 (Table S1; Fig. 1).The prevalence of serotype 23A among all IPD increased from 4.2% (n = 34) in 2008 to 13.4% (n = 60) and 13.8% (n = 31) in 2019 and 2020, respectively.Serotype 23A was the top non-vaccine serotype causing IPD between 2008 and 2011 but was replaced by serotype 15A between 2012 and 2017.Subsequently, serotype 23A returned to the most common non-vaccine serotype in 2018 (Table S1) and became the most dominant IPD-causing pneumococcal serotype by 2020 (more prevalent than other vaccine serotypes).The incidence of serotype 23A causing IPD increased annually between 2008 and 2019 and then decreased in 2020 owing to the COVID-19 pandemic.In February 2020, the implementation of a mask-wearing policy in Taiwan resulted in a decrease in the incidence of IPD-causing serotype 23A in 2020.However, serotype 23A remained the predominant serotype causing IPD in 2020.Before 2014, the genotype of serotype 23A was ST338; however, this clone shifted to ST166 by 2018 (6).Therefore, we collected pneumococci from two tertiary medical centers in 2019 and 2021 to study the major circulating ST166 serotype 23A pneumococci, a PMEN3 clone variant, in Taiwan after 2019.
b For penicillin, we defined the criteria for non-meningitis as MIC < 2 mg/L, susceptible, and MIC > 4 mg/L, non-susceptible.c For ceftriaxone, we defined the criteria for meningitis as MIC < 0.5 mg/L, susceptible, and MIC > 1 mg/L, non-susceptible.

PBP variation of 23A-ST166 increases the β-lactam non-susceptibility for S. pneumoniae R6
Considering that the MIC of amoxicillin in the 23A-ST166 isolates increased to 8 mg/L (Table 2; Table S2) compared with the MIC 0.5 mg/L of amoxicillin for 9V-ST156 and 9V-ST166 collected from 2001 and 2002 in Taiwan, we analyzed the effect of PBP variants on antimicrobial drug susceptibility of β-lactam, using amoxicillin as a selection antibiotic.The genomic DNA of 23A-ST166, with the highest MIC = 8 mg/L for amoxicillin in our study, was chosen as the PCR template.The MIC of R6−2X transformants for penicillin, amoxicillin, and meropenem increased by two-to four-fold, while largely increasing by 16-fold, from 0.03 mg/L to 0.5 mg/L for ceftriaxone.The MIC of R6-2X-2B transformants for amoxicillin, meropenem, and penicillin increased by 8-, 16-, and 4-fold, respectively.The MIC of R6-2X-2B transformants for ceftriaxone did not change.The MICs of the four β-lactam antibacterial drugs for the R6-2X-2B-1A transformant increased 2-16-fold, which was similar or the same level as that of 23A-ST166 (Table 2).Considering that there was no colony formation, we could not evaluate the MICs for transformants of R6−1a, R6−2b, and R6−2x-1a (Table 2).DNA sequence analysis of transformants in each transformation step confirmed that all amino acid substitutions occurred in the PBP1a, PBP2b, and PBP2x transpeptidase domains in the recipient strain.

Other antimicrobial resistance genes and pilus determinants
Among the whole-genome sequenced 23A-ST166 isolates, all had ermB, conferring resistance to macrolide antibiotics, tetM, conferring resistance to tetracycline, and a folA mutation (I100L substitution) known to decrease bacterial trimethoprim susceptibility.Only 4 of the 50 isolates had a folP insertion (two codon insertions between bases 168 and 201), conferring resistance to sulfamethoxazole; one isolate had a gyrA mutation, conferring resistance to fluoroquinolone, and none carried mutations in parC and gyrB.In addition, all but one 23A-ST166 isolate had pilus determinants (rlrA islets) in its genome (Table S2).

Phylogenetic tree analysis of 23A-ST166 compared with its ancestor clone 9V-ST156
Using ATCC-700671 (9V-ST156, GenBank accession no: CP099641) as an outgroup, we constructed a whole-genome phylogenetic tree for the 23A-ST166, 9V-ST156, and 9V-ST166 isolates (collected from Taiwan) (Fig. 2A).Compared with the original 9V-ST156 from Spain, 9V-ST156 circulated in Taiwan recombined at 10 regions and at an additional eight regions to be 9V-ST166, before the use of PCV13.Compared with 9V-ST166, there were 27 specific recombination events in all 23A-ST166 isolates, including several genes that might contribute to pneumococci evolution: (i) the capsule polysaccharide (cps) locus, which was closely related to that of an 23A

Global dissemination of PMEN3 (Spain 9v -ST156) variant
To study how PMEN3 lineages have evolved globally (Fig. 3), we constructed a wholegenome phylogenetic tree.From the database, 63 WGS data of S. pneumoniae strains were downloaded.Among these, 13 11A-ST838 and 36 11A-ST6521 were in Europe, and 14 35B-ST156 were in the United States.PMEN3 isolates were divided into the following three clades: (i) all 35B-ST156 isolates clustered together with 9V-ST156, (ii) all 23A-ST166 isolates clustered together with 9V-ST166, and (iii) all serotype 11A clustered together (Fig. 2A).Compared with the original 9V-ST156 from Spain, 35B-ST156 and 11A-ST838/ ST6521 had 25 and 7 unique recombination events, respectively.Several recombination events related to evolution, including the bacterial serotype switch-related recombina tion in the CPS region (in all PMEN3 variants), antibiotic susceptibility-related recombi nation in genes such as pbp1a, pbp2b, pbp2x, murM, and folP conferring resistance to β-lactam and sulfamethoxazole, and recombination of transposase-related and phagerelated genes that increase the fitness of pneumococci to adapt to environmental stress, were observed in most PMEN3 variants (Table S7; Fig. 2A).

DISCUSSION
The increase in amoxicillin-and meropenem-resistant serotype 23A replacement in Taiwan was mainly mediated by the expansion of non-vaccine serotype 23A with a previously well-adapted, globally spreading antibiotic-resistant PMEN3 lineage through multiple recombination events.The 9V-ST156 and its single-locus variant ST166 had been circulating before PCV13 in Taiwan (3,4).Serotype/capsular switch events in 23A-ST166 should have occurred between the 23A-ST338 strain as the serotype/capsular cps23A donor and the 9V-ST166 strain as the recipient.Genomic surveillance of the dynamics of pneumococcal populations after the widespread use of PCV revealed that previously existing clones changed their capsular types to non-vaccine types to evade vaccine-induced immunity, continuing their spread in the community (24).These previously existing successful clones usually harbor specific virulence factors or antibiotic resistance genes to be disseminated in many countries.Intriguingly, they are adept at undergoing capsular switching events to express various capsular types.For instance, the Spain 23F -1 clone (ST81) possesses an integrative conjugative element (ICE), MM1 phage, Na + -dependent ATPase Island, and TprA2/PhrA2 genomic regions, which are associated with increased colonization and virulence (25,26).ST81 also expresses alternative types, such as 19F, 14, 6B, and 15B (27).From 2013 to 2017, we reported that the Spain 23F -ST81 clone rapidly recombined in 12 regions and transformed into 15B/C-ST83, which became the most common penicillin-and merope nem-resistant clone in Taiwan (2).The expansion of successful clones mediated by capsular switching to non-vaccine types with high-level antibiotic resistance raises concerns regarding the historical predominance of these epidemic clones (15).
Similarly, the PMEN3 lineage in Taiwan acquired genetic variation through recombina tion with 23A-ST338 in the capsular synthesis region, 19A-ST320 on pbp2b, 23F-ST81 on xpt, and other multiple loci, such as pcpA, comF, and potD, which are associated with pneumococcal metabolism, virulence, natural competence, and antibiotic resistance development (28)(29)(30) for rapid adaptation to selective pressure.PBP2b-11, which originated from a pandemic multidrug-resistant 19A-ST320 clone, increased amoxicillin resistance (15) and had 10 unique changes in the 590-641 amino acid region, the key alteration associated with amoxicillin resistance (31,32).PMEN3 was initially suscepti ble to amoxicillin and non-susceptible to meropenem, turning into amoxicillin-and meropenem-resistant strains after receiving pbp2b from 19A-ST320.We also demonstra ted that the novel PBP2x-299 substantially increases the MIC of ceftriaxone.In a study of isolates belonging to the PMEN3 lineage from 31 countries between 1992 and 2012, a high frequency of recombination (r/m ratio:0.115recombination/point mutation) and great diversity of the imported sequence (11.8 single nucleotide polymorphisms/kb of sequence imported) were found in the PMEN3 reconstruction, which accounted for their successful dissemination between countries (33).In the late PCV13 period, together with global descendants that emerged after 2015, including 35B-ST156 in the United States and 11A-ST838/ST6521 in Europe, the global landscape of PMEN3 lineages involved repeated, selectively favored convergent recombination at CPS, PBPs, murM, and folP genome sites.These results were consistent with those of a previous study that demonstrated that the most common recombination events occurred on PMEN3 located in the capsular region (adjacent to pbp1a and pbp2x), murM, and pbp2b over a 30-year period (12).
The extent of serotype replacement by non-vaccine serotypes varies between countries.Non-PCV13 serotypes such as 11A, 15A, 35B, and 23A were prevalent in Asian countries (34); 12F, 15B/C, 24, and 5 in Israel (35); 8, 35B/D, 12F, 16F, and 15B/C in South Africa (35); and 15B/C, 22F, 33F, and 35B/D in the United States (35).Fur thermore, with selection by community antibiotic consumption, non-vaccine serotype pneumococci adaptively evolved with increased MIC of third-generation cephalosporins, fluoroquinolones, and carbapenems after the introduction of PCV13 (36).The emergence of multidrug-resistant pneumococci, a worrisome challenge for clinicians, may cause infections that fail to respond to commonly used antibiotics, resulting in life-threatening diseases.Serotype 23A has a low invasive disease potential, yet it frequently causes disease in immunocompromised hosts and individuals with chronic medical conditions (35).In recent years, an increase in serotype 23A has occurred not only in Asian countries (35) but also in Israel and Belgium (37,38).Currently, 23 valent pneumococcal polysaccharide vaccines (PPV23), PCV13, PCV15, and PCV20 include serotype 23F but not serotype 23A.Genes for capsular polysaccharide synthesis between serotypes 23A and 23F are similar, except for the oligosaccharide polymerase Wzy, which results in different polymerization and divergent polysaccharide structures (39).Although typing antiserum with serotype 23F reacts slightly with serotype 23A, immunity generated after pneumococcal vaccine containing serotype 23F cannot cross-protect against serotype 23A infection.Optimization of vaccination strategies using a PCV containing serotype 23A could be of value for patients with a high risk of serotype 23A infection.
In this study, we delineated the molecular mechanism of clonal shift within serotype 23A serotype replacement in Taiwan, deconvoluted the development of the ST166 lineage evolved from its ancestral clone, and analyzed genetic variation in PMEN3' descendants in other countries.These findings expand the current knowledge regard ing the remarkable evolutionary capacity of the PMEN3 lineage.In addition, we used a transformation study to demonstrate that the new PBP2x-299 contributed to the increased MIC of ceftriaxone in S. pneumoniae, presumably due to the additional eight aa changes that reduce the binding affinity of ceftriaxone to PBP2x.The limitations of this study include the following: (i) we collected serotype 23A isolates from only two medical centers, which may not be sufficiently representative.However, these isolates clustered tightly and exhibited consistent recombination sites.Additional genomic differences among isolates from various regions may be limited and (ii) all isolates were obtained from non-invasive sites.It is not clear whether the most invasive serotype 23A isolates belonged to ST166.Colonization is the first step in the development of pneumococcal disease.We believe that a clonal shift from ST338 to ST166 also occurred among the invasive isolates following the use of antibiotics and PCV13.
To date, the PCV strategy has been successful in decreasing the virulent capsular type without the selection of dangerous clones.Nevertheless, the convergence of extensive resistance through multiple recombination events in pre-existing epidemic clones of S. pneumoniae represents an alarming threat.Restriction of antibiotic overuse to inhibit the spread of super-resistant clones, especially those causing disease in patients with chronic disease, remains an important matter of concern.tion, Project administration, Resources, Supervision, Writing -original draft, Writingreview and editing

FIG 1
FIG 1 Incidence (lines) of IPD and proportions (bars) of IPD cases caused by serotype 23A during 2008-2020 post PCV13 immunization program in Taiwan (red triangle in 2011).Surveillance data from Taiwan Centers for Disease Control IPD Surveillance System show that the incidence (orange line) and percentage (orange bar) of serotype 23A increased during 2008-2020.Post PCV13 vaccination, the non-vaccine serotype with the highest incidence was serotype 23A between 2018 and 2020 ( # P = 0.011 in 2019 and *P = 0.05 in 2020, compared with 2008).In contrast, the total incidence of IPD (blue line) significantly decreased between 2013 and 2019 (**P < 0.001, compared with 2008).The black numbers indicate the numbers of IPD-causing serotype 23A.IPD, invasive pneumococcal disease; PCV13, 13-valent pneumococcal conjugate vaccine.
applicable; TW, Taiwan; ATCC, American Type Culture Collection; ND, not done; PBP, penicillin-binding protein; +, positive for colony formation on amoxicillin agar plates within 24 h of incubation; −, negative for colony formation on amoxicillin agar plates within 24 h of incubation.

FIG 3
FIG 3 Dissemination of the Spain 9V -ST156 (PMEN3) variant clone following PCV13 vaccination.The map was created using the amCharts Pixel Map Generator.

TABLE 1
Antimicrobial activity of six antimicrobial agents against 50 Streptococcus pneumoniae 23A-ST166 between 2019 and 2021

TABLE 2
Penicillin-binding protein transformation study a