TABLE S1: Association of 19A subtypes with the era after introduction of PCV7 using a multivariable logistic regression analysis

Transcription

1 1 SUPPLEMENTAL MATERIAL TABLE S1: Association of 19A subtypes with the era after introduction of PCV7 using a multivariable logistic regression analysis Odds Ratio (95% confidence interval) p-value Subtype 19A I 19A II 19A III 1 (base) 6.2 ( ) 1.46 ( ).5.4 Age of patient -1 years 2-4 years 5-15 years > 15 years 1 (base).59 ( ).38 ( ) 1.6 ( ) Female gender 1.71 ( ).17 Antimicrobial resistance Penicillin (.6 mg/l) Erythromycin Sulfamethoxazole/trimethoprim 2.15 ( ).39 ( ).54 ( ) (SXT) Geographical region East Switzerland West Switzerland 1 (base).72 ( ).43 Logistic regression model adjusted adjusted for potential confounders like age (-1 (base), 2-4, 5-15 and >15 years), sex (male gender as base), penicillin resistance (susceptible chosen as base), erythromycin resistance (susceptible chosen as base), SXT resistance (susceptible chosen as base) and geographical origin (east Switzerland as base). Minimum inhibitory concentration (MIC) for penicillin non-susceptibility was.6 mg/l while for erythromycin and SXT the disk diffusion method was performed (intermediate and resistant were considered as non-susceptible. Adjusted odds ratios (aor) with 95% confidence intervals (95%CI) are shown.

2 FIGURE S1A. HPLC chromatograms of monosaccharide standards (glucose, rhamnose, mannosamine, glucosamine, galactose, galactosamine). HPLC chromatograms showing retention time of glucose, rhamnose, mannosamine (Man-N), glucosamine (Glc-N), galactose, and galactosamine (Gal-N) served as a template for peak identification. Please note that acetylated aminosugars (e.g., N-acetyl mannosamine) are deacetylated during hydrolysis with TFA (1) and therefore only appear as aminosugars (e.g., mannosamine) peak in the run. Chromatograms are stacked to faciliate comparison and rhamnose and glucose peaks are labeled, Y-axis fluorescence (FU). 23

3 FIGURE S1B. HPLC chromatograms of capsule extracts from serotype 19F strain B21.73 and its isogenic capsule knockout mutant (B21.73 cps). To assess the degree of contamination from cell wall components in our assay serotype 19F strain B21.73 and its isogenic capsule knockout mutant were grown in CDM supplemented with glucose and extraction procedure was performed identically for both of them. Chromatograms are stacked to faciliate comparison and mannosamine (Man-N), rhamnose (Rha) and glucose (Glc) peaks are labeled, Y-axis fluorescence (FU). Early peaks are derived from pneumococcal cell wall polysaccharide (CWPS) and consist of P-choline residues followed by aminosugars including glucosamine and muramic acid from peptidoglycan and galactosamine from CWPS as determined by chromatogram comparison with purified pneumococcal cell wall polysaccharide (Statens Serum Institute, Copenhagen, Denmark) and monosaccharide standards including N-acetyl muramic acid. It is to note that N-acetyl aminosugars are deacetylated during hydrolysis with TFA (1). As aminosugar recovery has

4 been reported to be only party satisfying with this method (2) and can results in the formation of disaccharides which is reflected by a peak at approximately 18 minutes, we therefore didn t assess aminosugars by HPLC and used HPLC as a screen for additional sugars indicating a change in capsule composition. In addition, separation conditions in this HPLC assay is optimized for the detection of neutral monosaccharides and therefore, aminosugar separation resolution might be impaired

5 FIGURE S2. 19A subtype capsule extracts from (19A-I) grown in CDM supplemented with mucin MG1 monosaccharides and (19A-II) in pooled human saliva. To mimic saccharide nutrients present in the natural human environment of S. pneumoniae, (19A-I) was grown in CDM supplemented with monosaccharides detected in human mucin (5.5 mm total concentration of the mucin building monosaccharides in ratios as determined for the salivary mucin MG1) and (19A-II) was grown in pooled sterile filtrated human saliva collected from 1 healthy volunteers. Chromatograms are stacked to facilitate comparison, Y-axis fluorescence (FU)

6 Data File: Bau4938 Probe #9, Strain CDM Glc (1%) Injection Volume (µl): 5/1 RT: E6 MS Bau Component Name RT Area Calculated Sample Found Amount Yes Rham ,312µg Yes Glc ,956µg

7 d:\daten29\...\proben\baustein\bau4938 # CDM Glc 1% 5/1 11/12/29 8:43:31 PM RT: E6 m/z= F: bau E6 bau91116 a m/z

8 d:\daten29\...\proben\baustein\bau4938 # CDM Glc 1% 5/1 11/12/29 8:43:31 PM RT: E6 m/z= F: bau E6 bau91116 a m/z 4.13E5 bau4938#1361 RT: AV: 1 SB: , T: + c Full ms [ ] 1.28E4 bau4938#1347 RT: AV: 1 SB: , T: + c Full ms [ ] FIGURE S3A. GC-MS of (19A-I) capsule extracts grown in CDM with 55 mm glucose. GC-MS was used to confirm the presence of rhamnose and glucose in capsule extracts as HPLC only allows the identification of peaks based on retention time comparison with standards. GC raw pattern is given in the top panel of the figure and peaks of interest and corresponding MS spectra are shown in the panels below. MS spectra of glucose and rhamnose standards are shown in Figure S3F. 74

9 Data File: Bau4939 Probe #1, Strain PIM (1%) Injection Volume (µl): 5/1 RT: E6 MS Bau Component Found Name RT Area Calculated Amount (pmol) Sample Yes Rham ,354µg Yes Glc ,199µg

10 d:\daten29\...\proben\baustein\bau4939 # PIM 1% 5/1 11/12/29 9:13:32 PM RT: E6 m/z= F: bau E6 bau91116 a m/z FIGURE S3B. GC-MS of (19A-I) capsule extracts grown in PIM. GC-MS was used to confirm the presence of rhamnose and glucose in capsule extracts as HPLC only allows the identification of peaks based on retention time comparison with standards. GC raw pattern is given in the top panel of the figure and peaks of interest and corresponding MS spectra are shown in the lower panel. MS spectra of glucose and rhamnose standards are shown in Figure S3F. 89

11 Data File: Bau493 Probe #1, Strain 51.24PIM (1%) Injection Volume (µl): 5/1 RT: E7 MS Bau Component Name RT Area Calculated Sample Found Amount Yes Rham ,81µg Yes Glc ,868µg

12 D:\Daten29\...\Proben\Baustein\Bau493 # Pim 1% 5/1 11/12/29 4:43:28 PM RT: RT: 1.49 AA: RT: 1.63 AA: RT: 1.75 AA: RT: 1.9 AA: RT: 1.99 AA: RT: 11.8 AA: RT: 11.3 RT: AA: AA: E7 ICIS Bau E6 bau91116 a m/z 3.64E5 Bau493#895 RT: 1.9 AV: 1 T: + c Full ms [ ] 3.88E5 Bau493#867 RT: 1.75 AV: 1 T: + c Full ms [ ] 4.62E4 Bau493#3 RT: 1.67 AV: 1 T: + c Full ms [ ] 1.33E6 Bau493#842 RT: 1.62 AV: 1 T: + c Full ms [ ]

13 D:\Daten29\...\Proben\Baustein\Bau493 # Pim 1% 5/1 11/12/29 4:43:28 PM RT: RT: AA: RT: AA: RT: AA: RT: AA: RT: AA: RT: AA: 122 RT: 14.3 AA: RT: AA: RT: AA: E7 ICIS Bau E6 bau91116 a FIGURE S3C. GC-MS (19A-I) of capsule extracts grown in PIM. GC-MS was used to confirm the presence of rhamnose and glucose in capsule extracts as HPLC only allows the identification of peaks based on retention time comparison with standards. GC raw pattern is given in the top panel of the figure and peaks of interest and corresponding MS spectra are shown in the panels below. MS spectra of glucose and rhamnose standards are shown in Figure S3F m/z 6.62E4 Bau493#14 RT: AV: 1 T: + c Full ms [ ] 1.3E6 Bau493#1377 RT: AV: 1 T: + c Full ms [ ] 1.21E5 Bau493#1361 RT: AV: 1 T: + c Full ms [ ] 17

14 Data File: Bau4933 Probe #4, Strain ATCC 19A (1%) Injection Volume (µl): 5/1 RT: E6 MS Bau Component Found Name RT Area Calculated Amount (pmol) Sample Yes Rham ,35µg Yes Glc ,76µg

15 d:\daten29\...\proben\baustein\bau4933 #4 ATCC 19A 1% 5/1 11/12/29 6:13:25 PM RT: RT: 1.12 AA: RT: 1.27 AA: 4442 RT: 1.32 AA: RT: 1.37 AA: RT: 1.49 AA: RT: 1.62 AA: RT: 1.78 AA: RT: 1.91 RT: 1.99 AA: AA: RT: AA: E6 m/z= F: ICIS bau E6 bau91116 a4 bau4933 #834 RT: 1.62 AV: E5 T: + c Full ms [ ] m/z

16 d:\daten29\...\proben\baustein\bau4933 #4 ATCC 19A 1% 5/1 11/12/29 6:13:25 PM RT: RT: AA: 1588 RT: AA: RT: RT: RT: 13.4 AA: AA: AA: RT: AA: RT: AA: RT: AA: RT: AA: 4529 RT: AA: 6538 RT: 14.3 AA: 578 RT: 14.9 AA: E6 m/z= F: ICIS bau E6 bau91116 a4 bau4933 #834 RT: 1.62 AV: E5 T: + c Full ms [ ] m/z FIGURE S3D. GC-MS of ATCC purified pneumococcal capsule polysaccharide 19A. GC-MS was used to confirm the presence of rhamnose and glucose in capsule extracts as HPLC only allows the identification of peaks based on retention time comparison with standards. GC raw pattern is given in the top panel of the figure and peaks of interest and corresponding MS spectra are shown in the panels below. MS spectra of glucose and rhamnose standards are shown in Figure S3F. 123

17 Data File: Bau4932 Probe #3, Strain Saliva (1%) Injection Volume (µl): 5/1 RT: E6 MS Bau Component Found Name RT Area Calculated Amount (pmol) Sample Yes Rham ,534µg Yes Glc ,545µg

18 D:\Daten29\...\Proben\Baustein\Bau4932 # Saliva 1% 5/1 11/12/29 5:43:27 PM RT: RT: 1.14 AA: RT: 1.24 AA: RT: 1.32 AA: RT: 1.61 AA: RT: 1.38 AA: RT: 1.51 AA: RT: 1.75 AA: RT: 1.83 AA: RT: 1.9 AA: RT: 11.1 RT: 11.8 AA: AA: E5 ICIS Bau E6 bau91116 a Bau4932#783 RT: 1.32 AV: 1 T: + c Full ms [ ] m/z 4.67E4 Bau4932#887 RT: 1.9 AV: 1 T: + c Full ms [ ] 5.35E4 Bau4932#861 RT: 1.75 AV: 1 T: + c Full ms [ ] 2.22E4 Bau4932#845 RT: 1.67 AV: 1 T: + c Full ms [ ] E Bau4932#835 RT: 1.61 AV: 1 T: + c Full ms [ ] E4 Bau4932#816 RT: 1.51 AV: 1 T: + c Full ms [ ] 6.5E4

19 D:\Daten29\...\Proben\Baustein\Bau4932 # Saliva 1% 5/1 11/12/29 5:43:27 PM RT: RT: 13.6 RT: AA: AA: RT: AA: RT: AA: RT: AA: RT: AA: RT: AA: RT: 13.7 AA: RT: RT: AA: 5197 AA: RT: AA: RT: 14.3 AA: E6 ICIS Bau E6 bau91116 a m/z 2.18E4 Bau4932#13 RT: 13.7 AV: 1 T: + c Full ms [ ] 8.59E4 Bau4932#1358 RT: AV: 1 T: + c Full ms [ ] 1.96E4 Bau4932#1347 RT: AV: 1 T: + c Full ms [ ] FIGURE S3E. GC-MS of (19A-I) capsule extracts grown in pooled human saliva. GC-MS was used to confirm the presence of rhamnose and glucose in capsule extracts as HPLC only allows the identification of peaks based on retention time comparison with standards. GC raw pattern is given in the top panel of the figure and peaks of interest and corresponding MS spectra are shown in the panels below. MS spectra of glucose and rhamnose standards are shown in Figure S3F. 14

20 Bau91116a7 #879 RT: 1.6 AV: E5 T: + c Full ms [ ] m/z rhamnose Bau91116a7 #1425 RT: AV: E5 T: + c Full ms [ ] glucose FIGURE S3F. MS spectra of monosaccharide standards (rhamnose and glucose). MS spectra of glucose and rhamnose served as a template for peak identification (see Figures S3A-E). m/z

21 FIGURE S4. 2D NMR. Shown is a superimposition of 1 H- 13 C HSQC-NMR spectra of PS from Hungary 19A-6 capsule grown in CDM and PIM.

22 15 REFERENCES: Kwon H, Kim J Determination of monosaccharides in glycoproteins by reverse-phase high-performance liquid chromatography. Anal Biochem 215: Talaga P, Vialle S, Moreau M. 22. Development of a high-performance anion exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates. Vaccine 2: