We included 88 clinical S. pneumoniae isolates, 19 S. pseudopneumoniae isolates, 33 S. mitis isolates, 25 S. oralis isolates and 1 S. infantis isolate based on available WGS data and phenotypic susceptibility (MIC) for penicillin and for most isolates ceftriaxone (CFT). Isolates were retrieved from Danish and international collections of MGS (Supplementary Table S1): (1) Danish laboratory surveillance system at the Danish national Neisseria and Streptococcus Reference Laboratory (NSR), Statens Serum Institut, Denmark (SSI) (Kavalari et al., 2019), (2) Department of Biomedicine, Faculty of Health, Aarhus University, Denmark (Aarhus) (Jensen et al., 2015, 2016), (3) The Regional Department of Clinical Microbiology, Region Zealand, Slagelse Hospital, Denmark (Slagelse) (Rasmussen et al., 2016), (4) Queen Elizabeth II Health Sciences Center, Halifax, Canada via the Centers for Disease Control and Prevention, Atlanta, GA, United States (CDC) (Arbique et al., 2004), and (5) the One Day in Denmark (ODiD) project, National Food Institute, Technical University of Denmark, Denmark (Rebelo et al., 2022).

Isolates from SSI and the 10 S. pseudopneumoniae isolates from CDC were sequenced by paired-end Illumina sequencing (Illumina MiSeq) as previously described (Kavalari et al., 2019). For some isolates from Slagelse and all isolates from Aarhus, WGS data were retrieved from GenBank.¹ The remaining isolates from Slagelse were sequenced using Illumina HiSeq 2000 as previously described (4). Isolates from the ODiD-project were sequenced using the Illumina NextSeq 500 platform and pair-end sequencing and The Center for Genomic Epidemiology pipeline (Rebelo et al., 2022). Species identification was confirmed by cgMLSA from WGS data (Jensen et al., 2021). Software Pathogenwatch (Wellcome Sanger Institute)² was used for MLST and molecular serotyping of S. pneumoniae isolates. For MLST, the seven housekeeping genes aroE, gdh, gki, recP, spi, xpt and ddl were used, retrieved from the PubMLST website https://pubmlst.org/spneumoniae/ (Jolley et al., 2018). The method used for serotyping is based on SeroBA (Epping et al., 2018).

For the isolates from Slagelse/CDC, the ODiD-project and from SSI from year 2010, the MIC for penicillin and ceftriaxone was determined using Sensititre broth microdilution method (Streptococcus species MIC Plate, STP6F, Trek Diagnostic System, United Kingdom) according to manufacturer’s instructions. Before year 2010, the isolates from SSI was tested with Etest (AB Biodisk, Solna, Sweden) on Danish Blood Agar (Resistance plates, SSI Diagnostica) incubated at 36° C, 5% CO₂ (DANMAP, 2009). Isolates from Aarhus, were tested with agar dilution method (Jensen et al., 2015). EUCAST breakpoints table version 10³ was used for interpretation of SIR susceptibility.

Genotypic prediction of β-lactam susceptibility was performed using the classification system described by Li et al. (2016). From the CDC Streptococcus laboratory website,⁴ we obtained the amino acid sequences for the TPD subtypes of PBP1a, PBP2b and PBP2x in S. pneumoniae which at the time of data analysis was 101, 121, and 203 subtypes of PBP1a, PBP2b and PBP2x (June 2017). Using WGS data from our isolates, a nucleotide-protein BLAST analysis was performed using the NCBI Genome Workbench, version 3.0.0.⁵ to identify the nearest PBP1a, PBP2b and PBP2x subtypes. In the case of 100% identity for all three PBPs, a “PBP-profile” (PBP-type) was assigned, and the corresponding phenotypic MIC values for penicillin and ceftriaxone was retrieved from the available “PBP-type-To-MIC table,” accessed in June 2017. This contains 422 PBP-profiles for penicillin and 317 PBP-profiles for ceftriaxone. Although the number of PBP subtypes has increased significantly since (477 subtypes of PBP1a, 658 subtypes of PBP2b and 1050 subtypes of PBP2x by September 2022), the correlation of a whole PBP-type to a phenotypic susceptibility has not been updated since.

Isolates without a recognized PBP-profile included isolates where the exact combination of the three PBP-TPBs was not in the PBP-to-MIC table. Other isolates, particularly non-S. pneumoniae MGS, had substitutions in one or more PBP-TPDs. For MIC-prediction in these isolates, we created a database containing the concatenated PBP-TPD sequence for all published PBP-profiles with a correlating MIC. This database was used for a BLAST analysis with the concatenated PBP-TPD sequence for a given isolate. The best matching PBP-profile was identified as the result with the highest percent identity together with the highest total BLAST score.

Using this method, isolates could have a PBP-profile as best match with more substitutions than the sum of substitutions in each PBP separately. The applied method was chosen, because it requires only basic bioinformatic skills, a simple database containing the PBP-TPD amino acid sequences and the correlating phenotypic MIC and is fast to run.

A phylogenetic analysis of the concatenated PBP amino acid sequence from each isolate was performed using IQ-TREE (Nguyen et al., 2015) with a Blosum62 scoring matrix for amino-acid substitutions. The model only allowed amino acid sequences of equal length. All, except three S. mitis isolates, had a concatenated sequence of 914 amino acids. Isolates Sm7 and Sm19 had one insertion (position 326) and Sm32 three insertions (position 326–328), which were omitted for the phylogenetic analyses after ensuring that the PBP-profile was the same before and after the modification. We included the concatenated PBP amino acid sequence of the species type strains: S. pneumoniae NCTC 7465^T (NCBI reference NZ_LN831051.1), S. pseudopneumoniae ATCC-BAA-960^T (NZ_AICS00000000.1), S. mitis NCTC 12261^T (NZ_CP028414.1), S. oralis ATCC 35037^T (NZ_LR134336.1) and S. infantis ATCC 700779^T (NZ_GL732439.1). An outlier reference sequence was included: the concatenated PBP-sequence of Streptococcus dysgalactiae subspecies equisimilis AC2713 (NC_019042.1) was modified to a length of 914 amino acids by using an alignment with S. pneumoniae R6 to omit insertions at positions 525, 526, 532, 533, 650, 692, 897, and deletions at positions 327, 328, 867 were replaced by the corresponding amino acid in R6. The software iTOL version 5.6.2⁶ was used for visualization of trees.

We reported the number of isolates phenotypic and genotypic susceptible (S), susceptible-increased-exposure (I) and resistant (R) toward penicillin and ceftriaxone. Category agreement (CA) was defined as correctly predicted S-I-R, minor discrepancy (MiD) was susceptible isolates predicted to be susceptible-increased-exposure, susceptible-increased-exposure isolates predicted to be resistant and opposite. Major discrepancy (MaD) was isolates being resistant by genotype but susceptible by phenotype, while very major discrepancy (VMaD) was isolates being susceptible by genotype but resistant by phenotype. Essential agreement (EA) was an equal genotypic/ phenotypic MIC +/− one two-fold dilution. For isolates with an MIC ≤0.03, the value 0.03 was used.

For S. pneumoniae positive and negative predictive values (PPV, NPV) for non-susceptibility were calculated. Deming regression models were used to visualize log2 transformed phenotypic-genotypic MIC, using statistical software RStudio version 1.1.453. Isolates with an MIC ≤0.03, was given the value 0.03 and MIC >2 the value 4.

The data used did not include any personalized data.

In 55 of 88 S. pneumoniae isolates (62.5%), PBP1a, PBP2b and PBP2x all exactly matched a subtype in the database, and a recognized PBP-profile and a corresponding MIC could be predicted. Among isolates from the ODiD-project, 11 of 16 isolates (68.8%) had a recognized PBP-profile.

In the remaining 33 S. pneumoniae isolates, we found a new PBP-profile, since that exact combination of PBP-subtypes was not in the database, or there were new substitutions in the PBPs. There was between 1 and 43 substitutions in the concatenated PBP-sequence, when compared to the best matching recognized PBP-profile. (Table 1). Among the 88 S. pneumoniae isolates, there were 37 unique PBP-profiles, of which 17 were a recognized PBP-profile.

None of the non-S. pneumoniae MGS had a recognized PBP-profile. In S. pseudopneumoniae, 5 of 19 isolates had a PBP2b sequence also found in S. pneumoniae, (PBP2b0). Nearest PBP-profiles had 11–19 substitutions in the concatenated PBP-sequence (Supplementary Table S2). The 33 S. mitis isolates were more diverse in their PBP profiles with no isolate having the exact same amino acid sequence as another isolate. Five isolates had a PBP2b and/or PBP2x-subtype that were recognized from S. pneumoniae (Supplementary Table S4). The S. oralis isolates generally had more substitutions (40–91) than S. mitis. In 22 of the 25 isolates, the nearest PBP-profile was PT_17–1-22, but mostly with different substitutions (Supplementary Table S6). The one S. infantis isolate had 94 substitutions (Supplementary Table S8).

For six unique PBP-profiles, isolates had several MLSTs, while for six other unique PBP-profiles all isolates with the same profile had the same MLST. Isolates with the same MLST profile could have different PBP-profiles, e.g., ST-162 isolates had both PT_0–0-28 (8 substitutions, 9 isolates) and PT_78–0-0- (7 substitutions, 7 isolates) as the nearest PBP-profile. Most isolates belonged to serogroup 24 but represented eight different PBP-profiles. Likewise, PT_0–0-0 and PT_2–0-0- were seen in more than one serotype (Table 1).

Among the 55 S. pneumoniae isolates with a recognized PBP-profile, 46 were phenotypic and genotypic susceptible to penicillin, seven were phenotypic and genotypic susceptible-increased-exposure, and two were phenotypic and genotypic resistant, and this resulted in 100% CA (Table 2). EA was 98.2%; one isolate with a genotypic MIC of 2 μg/mL had a phenotypic MIC of 0.5 μg/mL (Table 3). Among the 33 isolates with a new PBP-profile not included in the database, more isolates had reduced susceptibility. CA was 90.9% with a MiD in two isolates and a MaD in one isolate (Table 2), and these three isolates contained many substitutions (23, 26, and 43). EA was 93.9% (Table 4). PPV and NPV for non-susceptibility were 93 and 94%. Genotypic-phenotypic accuracy using Deming regression showed a slope closer to unity for isolates with a recognized PBP-profile [1.01 (0.91–1.13)], compared to isolates with a new PBP-profile [1.04 (0.91–1.15)] (Figures 1A,B).

All S. pseudopneumoniae isolates were phenotypic and genotypic susceptible toward penicillin and CA was 100%. Among S. mitis isolates of which 27 were phenotypic susceptible, CA was only 75.8% with six MiD, one MaD, and one VMaD. Among S. oralis, two isolates were phenotypically resistant toward penicillin; one was resistant and one was susceptible-increased-exposure by genotype. The remaining isolates were phenotypically susceptible, but all except one was susceptible-increased-exposure by genotypic prediction, resulting in a very poor CA of 8% (2 of 25) (Table 2). All phenotypic and genotypic MICs for penicillin are presented in Supplementary Tables S3, S5, S7 and S9. Compared to S. pneumoniae, the non-S. pneumoniae MGS had much poorer MIC correlation in Deming regression curves (Figures 1C–E).

For S. pneumoniae isolates with a recognized PBP-profile, 52 isolates were genotypic susceptible, but one isolate with a genotypic MIC of ≤0.5 μg/mL had a phenotypic MIC of 1 μg/mL resulting in one MiD (1.8%) and thus a CA of 98.2%. This was however a single dilution difference. For isolates with a new PBP-profile, one isolate had no available genotypic MIC in the CDC PBP-to-MIC database. CA for S. pneumoniae isolates was 93.8%, and two isolates had a MiD. PPV and NPV for non-susceptibility was 100 and 98%. S. pseudopneumoniae isolates with an available susceptibility had a CA of 100%. In S. mitis, 30 isolates were phenotypic susceptible toward ceftriaxone, and three isolates had reduced phenotypic susceptibility. CA was 86.2% with three MaD and one VMaD. In S. oralis, two isolates were both phenotypic and genotypic resistant toward ceftriaxone, and the remaining isolates were genotypic and phenotypic susceptible with a 100% CA (Table 2). Genotypic-phenotypic MIC correlation for ceftriaxone is shown in Figures 1F–J.

We explored whether identifying the PBP subtype of only one of three PBPs in an isolate, could be enough to predict susceptibility (S, I or R), and this was the case for many subtypes. However, both susceptible and susceptible-increased-exposure isolates had common subtypes, such as PBP2b0, PBP2x0 or PBP2x2. For PBP1a only a few subtypes always predicted high level resistance, while other subtypes could be either I or R, or even S or R for one subtype (Supplementary Table S10). Therefore, it is necessary to identify the subtype of all three PBPs to predict susceptibility correctly.

For S. pneumoniae, the concatenated PBP amino acid sequence of the susceptible isolates clustered with that of the S. pneumoniae reference genome (NTCT 7465). Phenotypic non-susceptible isolates clustered in one larger group including isolates being either susceptible-increased-exposure or resistant and in one smaller group, where one isolate, Pn20, was susceptible by genotype. This isolate had 23 substitutions to the nearest PBP-type, which may explain this finding. Only one unique profile (isolate, Pn76) was susceptible by phenotype, but resistant by genotype (MaD). Three isolates, all with PBP-profile PT_17–15-22, but with 0, 18, and 26 substitutions respectively, clustered together and had similar phenotypic MICs between 0.5 and 1 μg/mL, indicating no functional importance of these substitutions on susceptibility (Figure 2). When including all species in the analysis, the type strains for S. pneumoniae, S. pseudopneumoniae, S. mitis and S. oralis all clustered with susceptible isolates of that species. A separate cluster of susceptible S. mitis isolates was seen, and both S. mitis clusters had related non-susceptible S. pneumoniae isolates, suggesting a horizontal gene transfer between these two species. The one S. infantis isolate clustered with one S. mitis and one S. oralis isolate, both susceptible by phenotype, but susceptible-increased-exposure by genotype. A separate cluster included isolates of S. pneumoniae, S. mitis and S. oralis being susceptible-increased-exposure and resistant, and surprisingly, the S. infantis type strain was in this cluster (Figure 3).