Complement Resistance and Igm Binding to Non-Typeable Haemophilus Influenzae Is Not Altered

Complement resistance and IgM binding to non-typeable Haemophilus influenzae is not altered when ascending from the nasopharynx to the middle ears in children with otitis media

Medical Microbiology and Immunology,

Jeroen D. Langereis,1# Thijs M. A. van Dongen,2 Kim Stol,1 Roderick P. Venekamp,2,3

Anne G. M. Schilder,2,3,4 and Peter W. M. Hermans1

1Laboratory of Pediatric Infectious Diseases, Radboud University Medical Centre, Nijmegen, The Netherlands

2Department of Epidemiology, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands

3Department of Otorhinolaryngology, Division Surgical Specialities, University Medical Center Utrecht, Utrecht, The Netherlands

4 ENT Clinical Trials Programme, Ear Institute, University College London, London, United Kingdom

#Corresponding author:

Jeroen D. Langereis

Laboratory of Pediatric Infectious Diseases,

Radboud University Medical Centre, Internal Post 224, P.O. Box 9101,

6500 HB Nijmegen, The Netherlands. Tel.: +31 24 3667345; Fax: +31 24 3666352; E-mail address:

Supplementary Figure 1. Survival of NTHi R2866, NTHi 3655 and Rd in 1.25%, 2.5%, 5.0% or 10 % pooled normal human serum (NHS) for 1 hour at 37ºC.

Supplementary Figure 2. Binding of serum IgG, serum IgM or TEPC-15 to whole cell lysate or LOS of nasopharyngeal (NPS) and middle ear fluid (MEF) NTHi isolates with identical MLST type was determined by Western blot analysis (n=12).

Supplementary Table 1. PCR primers used in this study.

Target / Primers / Sequence / Reference
bexA / HI-1 / CGTTTGTATGATGTTGATCCAGAC / (14)
HI-2 / TGTCCATGTCTTCAAAATGATG / (14)
bexB / bexB.1F / GGTGATTAACGCGTTGCTTATGCG / (13)
bexB.1R / TTGTGCCTGTGCTGGAAGGTTATG / (13)
pepN / pepN-F / GATGGTCGCCATTGGGTGG / (10)
pepN-R / GATCTGCGGTTGGCGGTGTGG / (10)
iga / iga_F / GTTCCACCACCTGCGCCTGCTAC / (12)
iga_R / GTTATATTGCCCCTCGTTATTCA / (12)
fucK / fucK_F / ACCACTTTCGGCGTGGATGG / (11)
fucK_R / AAGATTTCCCAGGTGCCAGA / (11)

Supplementary Table 2. MLST analysisof NTHi isolates isolated from the nasopharynx (NPS) or middle ear (MEF) of patients with rAOM, COME and ATTO. Genetically identical isolates used in the NPS-MEF correlations are depicted in bold.

Patient
number / Diagnosis / Isolate
number / Isolate
type / NTHi
PCR / MLST allele numbers / Sequence
Type
Adk / atpG / frdB / fucK / mdh / pgi / recA
C001 / rAOM / MEF_C001 / MEF / NT / 6 / 100 / 121 / 5 / 206 / 130 / 19 / 919
C001 / rAOM / NPS_C001 / NPS / NT / 35 / 25 / 16 / 53 / 7 / 40 / 10 / 1012
C004 / COME / MEF_C004 / MEF / NT / 1 / 1 / 1 / 1 / 67 / 1 / 5 / 367
C004 / COME / NPS_C004 / NPS / NT / No MLST
C006 / COME / MEF_C006 / MEF / NT / 14 / 7 / 13 / 7 / 17 / 13 / 17 / 57
C006 / COME / NPS_C006 / NPS / NT / 14 / 7 / 13 / 7 / 17 / 13 / 17 / 57
C008 / COME / MEF_C008 / MEF / NT / 11 / 2 / 15 / 8 / 28 / 26 / 3 / 34
C008 / COME / NPS_C008 / NPS / NT / No MLST
C009 / rAOM / MEF_C009 / MEF / NT / 1 / 1 / 1 / 14 / 89 / 127 / 37 / 914
C009 / rAOM / NPS_C009 / NPS / NT / 1 / 1 / 1 / 14 / 89 / 127 / 37 / 914
C013 / COME / MEF_C013 / MEF / NT / 40 / 57 / 22 / 43 / 7 / 70 / 119 / 920
C013 / COME / NPS_C013 / NPS / NT / 3 / 2 / 53 / 2 / 7 / 8 / 10 / 915
C049 / COME / MEF_C049 / MEF / NT / 28 / 33 / 7 / 18 / 11 / 125 / 89 / 478
C049 / COME / NPS_C049 / NPS / NT / 28 / 33 / 7 / 18 / 11 / 125 / 89 / 478
C062 / rAOM / MEF_C062 / MEF / NT / 14 / 7 / 13 / 15 / 17 / 13 / 1 / 98
C062 / rAOM / NPS_C062 / NPS / NT / 14 / 7 / 13 / 15 / 17 / 13 / 1 / 98
C089 / rAOM / MEF_C089 / MEF / NT / 16 / 8 / 16 / 16 / 30 / 1 / 3 / 155
C089 / rAOM / NPS_C089 / NPS / NT / 16 / 8 / 16 / 16 / 30 / 1 / 3 / 155
C101 / COME / MEF_C101 / MEF / NT / 5 / 33 / 7 / 15 / 22 / 1 / 29 / 544
C101 / COME / NPS_C101 / NPS / NT / No MLST
C109 / COME / MEF_C109 / MEF / NT / 5 / 8 / 58 / 14 / 71 / 13 / 29 / 276
C109 / COME / NPS_C109 / NPS / NT / 5 / 8 / 58 / 14 / 71 / 13 / 29 / 276
C115 / COME / MEF_C115 / MEF / NT / 63 / 8 / 58 / 8 / 92 / 58 / 29 / 355
C115 / COME / NPS_C115 / NPS / NT / 63 / 8 / 58 / 8 / 92 / 58 / 29 / 355
R006 / COME / MEF_R006 / MEF / NT / 40 / 1 / 1 / 14 / 1 / 59 / 3 / 160
R006 / COME / NPS_R006 / NPS / NT / 40 / 1 / 1 / 14 / 1 / 59 / 3 / 160
R015 / COME / MEF_R015 / MEF / NT / 63 / 57 / 52 / 7 / 26 / 130 / 21 / 916
R015 / COME / NPS_R015 / NPS / NT / 63 / 57 / 52 / 7 / 26 / 130 / 21 / 916
R020 / COME / MEF_R020 / MEF / NT / 107 / 20 / 7 / 12 / 26 / 92 / 29 / 589
R020 / COME / NPS_R020 / NPS / NT / 107 / 20 / 7 / 12 / 26 / 92 / 29 / 589
R021 / COME / MEF_R021 / MEF / NT / 33 / 8 / 8 / 16 / 1 / 2 / 3 / 201
R021 / COME / NPS_R021 / NPS / NT / 33 / 8 / 8 / 16 / 1 / 2 / 3 / 201
R033 / COME / MEF_R033 / MEF / NT / 1 / 3 / 1 / 1 / 31 / 1 / 5 / 40
R033 / COME / NPS_R033 / NPS / NT / 26 / 17 / 8 / 1 / 78 / 57 / 50 / 249
R035 / COME / MEF_R035 / MEF / NT / 14 / 7 / 1 / 15 / 10 / 4 / 1 / 917
R035 / COME / NPS_R035 / NPS / NT / 14 / 7 / 1 / 15 / 10 / 4 / 1 / 917
R038 / COME / MEF_R038 / MEF / NT / 69 / 55 / 10 / 36 / 79 / 82 / 56 / 272
R038 / COME / NPS_R038 / NPS / NT / 69 / 55 / 10 / 36 / 79 / 82 / 56 / 272
R047 / COME / MEF_R047 / MEF / NT / 1 / 1 / 1 / 35 / 15 / 53 / 38 / 142
R047 / COME / NPS_R047 / NPS / NT / 1 / 1 / 1 / 35 / 15 / 53 / 38 / 142
R048 / rAOM / MEF_R048 / MEF / NT / 1 / 1 / 1 / 35 / 15 / 53 / 89 / 142
R048 / rAOM / NPS_R048 / NPS / NT / 5 / 33 / 7 / 73 / 205 / 182 / 29 / 918
LOT 00388 / ATTO / 501669 / MEF / NT / 44 / 2 / 16 / 37 / 17 / 2 / 3 / 165
LOT 00388 / ATTO / 501670 / NPS / NT / 17 / 4 / 32 / 41 / 7 / 12 / 11 / 714
LOT 00416 / ATTO / 504158 / MEF / NT / 40 / 1 / 1 / 14 / 1 / 59 / 3 / 160
LOT 00416 / ATTO / 504159 / NPS / NT / 107 / 20 / 7 / 12 / 26 / 92 / 29 / 589
LOT 01493 / ATTO / 506487 / MEF / NT / 42 / 8 / 7 / 41 / 17 / 14 / 10 / 200
LOT 01493 / ATTO / 506488 / NPS / NT / 14 / 7 / 1 / 15 / 16 / 4 / 1 / 2
LOT00308 / ATTO / 510154 / MEF / NT / 42 / 57 / 57 / 14 / 7 / 72 / 4 / 410
LOT00308 / ATTO / 510155 / NPS / NT / 39 / 8 / 35 / 11 / 65 / 48 / 19 / 425
LOT 01460 / ATTO / 511633 / MEF / NT / 1 / 8 / 1 / 14 / 9 / 14 / 13 / 11
LOT 01460 / ATTO / 511634 / NPS / NT / 1 / 8 / 1 / 14 / 9 / 14 / 13 / 11
LOT 00162 / ATTO / 512949 / MEF / NT / 5 / 8 / 58 / 14 / 71 / 13 / 29 / 276
LOT 00162 / ATTO / 512950 / NPS / NT / 47 / 60 / 19 / 1 / 94 / 90 / 62 / 992
LOT 01024 / ATTO / 515119 / MEF / NT / 52 / 1 / 1 / 14 / 89 / 62 / 37 / 203
LOT 01024 / ATTO / 515128 / NPS / NT / 37 / 35 / 57 / 8 / 7 / 183 / 13 / 925
LOT 00810 / ATTO / 515496 / NPS / NT / 41 / 8 / 29 / 9 / 68 / 57 / 8 / 161
LOT 00810 / ATTO / 515497 / MEF / NT / 41 / 8 / 29 / 9 / 68 / 57 / 8 / 161
LOT 01527 / ATTO / 515613 / NPS / NT / 17 / 4 / 17 / 1 / 7 / 12 / 11 / 41
LOT 01527 / ATTO / 515614 / MEF / NT / 17 / 4 / 17 / 1 / 7 / 12 / 11 / 41
LOT 01107 / ATTO / 516302 / MEF / NT / 165 / 78 / 105 / 7 / 20 / 134 / 94 / 1215
LOT 01107 / ATTO / 516303 / NPS / NT / 165 / 78 / 105 / 7 / 20 / 134 / 94 / 1215
LOT 00673 / ATTO / 518804 / MEF / NT / 63 / 57 / 52 / 7 / 26 / 130 / 21 / 916
LOT 00673 / ATTO / 518805 / NPS / NT / 33 / 8 / 16 / 16 / 49 / 2 / 3 / 107
LOT 00481 / ATTO / 523019 / MEF / NT / 14 / 7 / 1 / 15 / 16 / 4 / 1 / 2
LOT 00481 / ATTO / 523020 / NPS / NT / 14 / 7 / 1 / 15 / 16 / 4 / 1 / 2
LOT 00903 / ATTO / 528612 / MEF / NT / 10 / 2 / 15 / 8 / 26 / 61 / 3 / 396
LOT 00903 / ATTO / 528613 / NPS / NT / 10 / 2 / 15 / 8 / 26 / 61 / 3 / 396
LOT 01170 / ATTO / 529674 / MEF / NT / 105 / 18 / 53 / 2 / 76 / 152 / 34 / 716
LOT 01170 / ATTO / 529675 / NPS / NT / 105 / 18 / 53 / 2 / 76 / 152 / 34 / 716
LOT 00476 / ATTO / 530428 / MEF / NT / 5 / 8 / 58 / 14 / 91 / 13 / 29 / 264
LOT 00476 / ATTO / 530429 / NPS / NT / 5 / 8 / 58 / 14 / 91 / 13 / 29 / 264
LOT00997 / ATTO / 533954 / NPS / NT / 14 / 7 / 13 / 7 / 17 / 13 / 17 / 57
LOT00997 / ATTO / 533958 / MEF / NT / 14 / 7 / 13 / 7 / 17 / 13 / 17 / 57
LOT 01022 / ATTO / 534113 / MEF / NT / 39 / 8 / 35 / 11 / 65 / 48 / 19 / 425
LOT 01022 / ATTO / 534114 / NPS / NT / 39 / 8 / 35 / 11 / 65 / 48 / 19 / 425
LOT00722 / ATTO / 542640 / MEF / NT / 14 / 7 / 1 / 15 / 16 / 4 / 1 / 2
LOT00722 / ATTO / 542642 / NPS / NT / 14 / 7 / 1 / 15 / 16 / 4 / 1 / 2
LOT 350 / ATTO / 543576 / MEF / NT / 3 / 54 / 65 / 1 / 64 / 78 / 48 / 474
LOT 350 / ATTO / 543577 / NPS / NT / 3 / 54 / 65 / 1 / 64 / 78 / 48 / 474
LOT 01180 / ATTO / 545128 / NPS / NT / 1 / 22 / 19 / 19 / 32 / 17 / 2 / 43
LOT 01180 / ATTO / 545129 / MEF / NT / 1 / 22 / 19 / 19 / 32 / 17 / 2 / 43
LOT 00797 / ATTO / 545769 / MEF / NT / 11 / 2 / 15 / 8 / 28 / 26 / 3 / 34
LOT 00797 / ATTO / 545771 / NPS / NT / 35 / 1 / 22 / 36 / 7 / 1 / 29 / 1216
LOT 00470 / ATTO / 548893 / NPS / NT / 113 / 33 / 7 / 1 / 10 / 41 / 29 / 942
LOT 00470 / ATTO / 548894 / MEF / NT / 113 / 33 / 7 / 1 / 10 / 41 / 29 / 942
LOT 01305 / ATTO / 549445 / MEF / NT / 16 / 8 / 16 / 16 / 30 / 1 / 3 / 155
LOT 01305 / ATTO / 549447 / NPS / NT / 16 / 8 / 16 / 16 / 30 / 1 / 3 / 155
LOT 00562 / ATTO / 563196 / MEF / NT / 42 / 2 / 38 / 41 / 7 / 55 / 10 / 1217
LOT 00562 / ATTO / 563197 / NPS / NT / 33 / 8 / 16 / 16 / 17 / 2 / 127 / 1218
LOT00576 / ATTO / 566792 / NPS / NT / 5 / 33 / 7 / 73 / 205 / 182 / 29 / 918
LOT00576 / ATTO / 566793 / MEF / NT / 5 / 33 / 7 / 73 / 205 / 182 / 29 / 918
LOT 01407 / ATTO / 572210 / NPS / NT / 14 / 7 / 1 / 15 / 16 / 4 / 1 / 2
LOT 01407 / ATTO / 572211 / MEF / NT / 14 / 7 / 13 / 7 / 17 / 13 / 17 / 57
LOT 01352 / ATTO / 576103 / MEF / NT / 113 / 33 / 7 / 1 / 210 / 41 / 29 / 1219
LOT 01352 / ATTO / 576104 / NPS / NT / 5 / 8 / 58 / 14 / 71 / 13 / 29 / 276
LOT 01395 / ATTO / 577099 / NPS / NT / 47 / 60 / 19 / 1 / 94 / 90 / 62 / 992
LOT 01395 / ATTO / 577100 / MEF / NT / 47 / 60 / 19 / 1 / 94 / 90 / 62 / 992
LOT 01091 / ATTO / 578649 / MEF / NT / 5 / 33 / 7 / 15 / 26 / 1 / 29 / 1220
LOT 01091 / ATTO / 578650 / NPS / NT / 5 / 33 / 7 / 15 / 26 / 1 / 29 / 1220
LOT 170 / ATTO / 579437 / MEF / NT / 63 / 32 / 7 / 18 / 46 / 74 / 29 / 1221
LOT 170 / ATTO / 579438 / NPS / NT / 5 / 33 / 7 / 15 / 22 / 1 / 29 / 544
LOT 185 / ATTO / 581879 / MEF / NT / 11 / 43 / 35 / 11 / 51 / 31 / 19 / 311
LOT 185 / ATTO / 581884 / NPS / NT / 11 / 43 / 35 / 11 / 51 / 31 / 19 / 311
LOT 01308 / ATTO / 584363 / MEF / NT / 23 / 3 / 5 / 1 / 11 / 66 / 21 / 760
LOT 01308 / ATTO / 584368 / NPS / NT / 5 / 33 / 7 / 15 / 47 / 58 / 29 / 176
LOT 00602 / ATTO / 587504 / MEF / NT / 5 / 1 / 1 / 1 / 1 / 2 / 5 / 14
LOT 00602 / ATTO / 587507 / NPS / NT / 5 / 1 / 1 / 1 / 1 / 2 / 5 / 14
LOT 00236 / ATTO / 587521 / MEF / NT / 17 / 4 / 32 / 41 / 7 / 12 / 11 / 714
LOT 00236 / ATTO / 587523 / NPS / NT / 17 / 4 / 32 / 41 / 7 / 12 / 11 / 714
LOT 00719 / ATTO / 587748 / MEF / NT / 33 / 117 / 16 / 16 / 17 / 142 / 29 / 1222
LOT 00719 / ATTO / 587749 / NPS / NT / 95 / 54 / 58 / 72 / 7 / 55 / 48 / 1223
LOT 00675 / ATTO / 588075 / MEF / NT / 161 / 43 / 70 / 20 / 62 / 217 / 129 / 1186
LOT 00675 / ATTO / 588076 / NPS / NT / 161 / 43 / 70 / 20 / 62 / 217 / 129 / 1186
LOT 00278 / ATTO / 590960 / MEF / NT / 51 / 36 / 1 / 81 / 158 / 38 / 18 / 832
LOT 00278 / ATTO / 590962 / NPS / NT / 51 / 36 / 1 / 81 / 158 / 38 / 18 / 832
LOT 01185 / ATTO / 590963 / NPS / NT / 42 / 9 / 8 / 2 / 158 / 8 / 4 / 1224
LOT 01185 / ATTO / 590964 / MEF / NT / 42 / 9 / 8 / 2 / 158 / 8 / 4 / 1224
LOT 00325 / ATTO / 605264 / NPS / NT / 1 / 22 / 19 / 19 / 32 / 17 / 2 / 43
LOT 00325 / ATTO / 605265 / MEF / NT / 1 / 22 / 19 / 19 / 32 / 17 / 2 / 43
LOT 348 / ATTO / 605446 / MEF / NT / 42 / 2 / 38 / 41 / 104 / 55 / 10 / 393
LOT 348 / ATTO / 605451 / NPS / NT / 42 / 2 / 38 / 41 / 104 / 55 / 10 / 393
LOT 347 / ATTO / 605460 / NPS / NT / 1 / 1 / 1 / 13 / 13 / 25 / 16 / 12
LOT 347 / ATTO / 605461 / MEF / NT / 1 / 1 / 1 / 13 / 13 / 25 / 16 / 12

ATTO; Acute tympanostomy tube otorrhea, rAOM; Recurrent acute otitis media, COME; Chronic otitis media with effusion, MEF; middle ear fluid, NPS; nasopharyngeal swab.