A new microarray system to detect Streptococcus pneumoniae serotypes.

Streptococcus pneumoniae, one of the most common gram-positive pathogens to colonize the human upper respiratory tract, is responsible for many severe infections, including meningitis and bacteremia. A 23-valent pneumococcal vaccine is available to protect against the 23 S. pneumoniae serotypes responsible for 90% of reported bacteremic infections. Unfortunately, current S. pneumoniae serotype testing requires a large panel of expensive antisera, assay results may be subjective, and serotype cross-reactions are common. For this study, we designed an oligonucleotide-based DNA microarray to identify glycosyltransferase gene sequences specific to each vaccine-related serotype. Out of 56 isolates representing different serotypes, only one isolate, representing serotype 23A, was not detected correctly as it could not be distinguished from serotype 23F. Our data suggest that the microarray provides a more cost-effective and reliable way of monitoring pneumococcal capsular types.

1. Introduction

Streptococcus pneumoniae is an important cause of bacteremia, community-acquired bacterial pneumonia, and meningitis, especially among young children and older adults [1-3]. Capsular polysaccharide is the primary S. pneumoniae virulence factor and encapsulated pneumococci are responsible for more diseases than unencapsulated strains [4]. After comparing the differences in capsular polysaccharides composition, S. pneumoniae can be divided into more than 90 serotypes [5] and the 23 serotypes responsible for 90% of disease cases [6] are represented in a 23-valent pneumococcal vaccine. Pneumococcal serogroup and serotype identification is currently performed by using large panels of expensive antisera by various methods, including the capsular swelling (Quellung) reaction, latex agglutination, and coagglutination. Cross-reactions between serotypes and discrepancies between methods can occur and some strains are nonserotypable. On the other hand, molecular typing has the potential to improve discrimination and provide additional information. S. pneumoniae capsule production is predominantly controlled by capsular polysaccharide synthesis (cps) gene clusters [7, 8], which are responsible for each serotype-specific polysaccharide. The Sanger Institute has sequenced the cps gene clusters of 90 S. pneumoniae serotypes and predicted the general function of 1,973 of the 1,999 gene products [9, 10]. S. pneumoniae capsular polysaccharides represent a diverse group of polymers with distinct sugar compositions and linkages [10]. The key enzymes to link each serotype-specific sugar component are glycosyltransferases (GTs) [11], which transfer the sugar moiety from an activated nucleotide sugar to an acceptor to generate a serotype-specific capsular polysaccharide. After discovering that S. pneumoniae GT genes are highly variable and contain serotype- or serogroup-specific regions, we used GT sequences as probes in an oligonucleotide-based microarray to identify 23-valent pneumococcal vaccine and closely related S. pneumoniae serotypes. Our data suggest that the microarray provides a more cost-effective and reliable way of monitoring serotype distribution.

2. Materials and Methods

2.1. Bacterial Strains, Growth Conditions, Immunological Serotyping, and Genomic DNA Extraction

S. pneumoniae strains representing various serotypes were obtained from the American Type Culture Collection, the Statens Serum Institute, and clinical isolates (Table 1). Each strain was cultivated on brain-heart infusion broth (Eiken, Tokyo, Japan) supplemented with 0.3% yeast extract (Becton Dickinson, Boston, MA) (BHI-Y) for 24 h at 37°C in 5% CO2. Conventional serotyping was performed for clinical isolates obtained in Japan by slide agglutination (Denka Seiken, Tokyo, Japan) or quellung reaction (Statens Serum Institute, Copenhagen, Denmark).

Table 1

Test strains.

Serotype	Strain designation	Serotype	Strain designation
1	ATCC6301a	14	D59b
2	ATCC6302a	15F	ATCC6315a
3	D36b	15A	ATCC6330a
4	JHK27b	15B	ATCC10354a
5	ATCC6305a	15C	SSI15C/2c
6A	MSC1943b	17F	ATCC6317a
6B	MSC1047b	17A	SSI17A/2c
7F	ATCC10351a	18F	ATCC6318a
7A	ATCC6307a	18A	ATCC10344a
7B	ATCC10348a	18B	ATCC10355a
7C	ATCC10350a	18C	ATCC10356a
8	ATCC6308a	19F	D33b
9A	ATCC8333a	19A	D4b
9V	KD10-11b	19B	ATCC10358a
9L	ATCC10349a	19C	ATCC10359a
9N	KD01-26b	20	ATCC6320a
10F	ATCC6310a	22F	KD01-23b
10A	ATCC8334a	22A	ATCC10363a
10B	SSI10B/2c	23F	KD11-15b
10C	SSI10C/2c	23A	KD12-06b
11F	ATCC6311a	23B	ATCC10364a
11A	SSI11A/2c	33F	ATCC10370a
11B	SSI11B/2c	33A	ATCC8340a
11C	ATCC10353a	33B	ATCC10342a
11D	SSI11D/1c	33C	ATCC8339a
12F	ATCC6312a	33D	SSI33D/2c
12A	SSI12A/5c	44	SSI44/3c
12B	SSI12B/1c	46	SSI46/2c

Explanatory notes: Serotypes represented in bold letter are those included in 23-valent pneumococcal vaccine.

aAmerican Type Culture Collection.

bClinical isolate obtained from Japan.

cStatens Serum Institute.

Genomic DNA was extracted using a Wizard Genomic DNA purification kit (Promega, Madison, WI).

2.2. DNA Array Preparation

Oligonucleotide probes were synthesized and spotted on a glass slide at Nihon Gaishi (Nagoya, Japan). The slide was stirred in a beaker filled with 2 × SSC/0.2% SDS for 15 min, transferred to a second beaker filled with 2 × SSC/0.2% SDS to incubate for 5 min at 95°C, rinsed three times with dH2O, and centrifuged at 900 rpm for 3 min at 25°C in a horizontal microtiter plate rotor before being covered with a plastic seal.

2.3. Chromosomal DNA Labeling

500 ng of genomic DNA was suspended in 21 μL dH2O and 20 μL of 2.5 × Random Primer Solution (Invitrogen, Carlsbad, CA), heated to 95°C for 5 min, and chilled on ice for 3 min. The DNA was labelled in a reaction including 5 μL of 10X dCTP Nucleotide Mix (Invitrogen, Carlsbad, CA), 5 μL Cy3 or Cy5-dCTP (GE Healthcare, Buckinghamshire, UK), and 1 μL of Exo-Klenow Fragment (Invitrogen, Carlsbad, CA ). After a 2-hour incubation at 37°C, 5 μL of sodium acetate, 125 μL of ethanol and 1 μL of glycogen was added to 25 μL of Cy3 and Cy5 labeled DNA, which was purified previously by QIAprep Spin Miniprep Kit (250) (Qiagen, Tokyo, Japan). Following a 30-minute incubation at −80°C in the dark, the probe mixture was centrifuged for 30 min at 14,000 rpm at 4°C. The supernatant was removed and the probe was air-dried for 5 min in the dark. The probe mixture was diluted in 70 μL of the hybridization buffer (25% formamide, 0.1% SDS, 6 × SSPE), incubated for 30 min at room temperature in the dark, heated for 8 min at 75°C, and incubated for 30 min at 42°C.

2.4. Probe Hybridization and Microarray Signal Detection

Prewarmed probe mixture was applied to the prepared microarray slide, placed in a hybridization chamber and incubated for 20 h at 42°C. After hybridization, the plastic seal was removed and the slide was washed with 1 × SSC/0.1% SDS solution for 3 min, 0.05 × SSC for 3 min, and 95% ethanol for 90 s at room temperature. The washed microarray slide was dried by centrifugation and scanned using the DNA Microarray Scanner (Agilent, Santa Clara, CA).

2.5. Data Analysis

The signal and background intensities of each spot were quantified using GenePix Pro 6.0 software and the average was calculated with Microsoft Excel software.

3. Results

3.1. Target Gene Selection and Microarray Construction

In this study, we designed a DNA microarray to identify the 23 S. pneumoniae serotypes included in the 23-valent pneumococcal vaccine, using GT genes in cps locus. We compared the GT sequences of the 23-valent vaccine serotypes with other S. pneumoniae serotypes and found that these 23 serotypes were indistinguishable from 14 nonvaccine serotypes. Therefore, 37 serotypes, 23-valent vaccine serotypes and 14 closely related serotypes, were divided into 23 groups and each group had one to six GT genes in their cps locus (Table 2). The 60-bp oligonucleotide probes contained the variable middle region of each open reading frame and were designed from published sequences at the Sanger Institute (http://www.sanger.ac.uk/Projects/S_pneumoniae/CPS/) and Genbank websites. In most cases, the designed probes were gene specific, although some probes included sequences from more than one gene. Each serotype group was identified using 3 to 18 probes (Table 2) and a total of 222 probes were designed to target 23 groups (Table 3). 26 positive control probes were designed to hybridize S. pneumoniae housekeeping genes and 16S rDNA. In addition, 26 negative control probes were designed to detect housekeeping genes of other bacterial respiratory pathogens, including Klebsiella pneumoniae, Staphylococcus aureus, Legionella pneumophila, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Pseudomonas aeruginosa, and Streptococcus pyogenes. A schematic diagram of the probe positions on the microarray is shown in Figure 1(a).

Table 2

Twenty-three groups distinguished in this study and targeted glycosyltransferase genes.

Group name	Targeted GT genes in cps locus (probe numbera)
1	wchB (1,2, 3)	wchD (4,5, 6)
2	wchF (7,8, 9)	wchG (10,11,12)	wchH (13,14,15)	wchI (16,17,18)
3	wchE (19,20,21)
4	wciJ (22,23,24)	wciK (25,26,27)	wciL (28,29,30)
5	wciJ (31,23,24)	whaC (32,33,34)	whaD (35,36,37)
6A/6B	wciN (38,39,40)	wciP (41,42,43)
7F/7A	wchF (44,45,46)	wcwA (47,48,49)	wcwF (50,51,52)	wcwG (53,54,55)	wcwH (56,57,58)
8	wciR (59,60,61)	wciR (62,63,64)	wciS (65,66,67)	wciT (68,69,70)
9A/9V	wchO (71,72,73,74)	wcjA (75,76,77)	wcjB (78,84,85)	WcjC (81,82,83)
9L/9N	wchO (71,72,73,74)	wcjA (75,76,77)	wcjB (78,79,80)	wcjC (81,82,83)
10A	wciB (86,87,8)	wcrC (89,90,91)	wcrD (92,93,94)	wciF (95,96,97)	wcrG (98,99,100)
11A/11D	wchK (101,102,103)	wcyK (104,105,106)	wcrL (107,108,109)
12F/12A/12B/44/46	wciJ (110,111,112)	wcxB (113,114,115)	wcxD (116,117,118)	wcxE (119,120,121)	wcxF (122,123,124)
14	wchK (125,126,127)	wchL (128,129,130,131)	wchM (132,133,1334)	wchN (135,136,137)
15B/15C	wchK (138,139,125)	wchL (128,140,141,131)	wchM (142,143)	wchN (135)
17F	wchF (144,145,146)	abp1 (147,148, 149)	wciP (150,151,152)	wcrV (153,154,155)
18B/18C	wchF (156,157)	wciU (158,159,160)	wciV (161,162,163)	wciW (164,165,166)
19F	wchO (167,72,168,169)	wchQ (171,172,173)
19A	wchO (71,170,73,74)	wchQ (171,172,173)
20	wciB (174,175,176)	whaJ (177,178,179)	wciL (180,181,182)	wcwK (183,184,185)	wciD (186,187,188)	whaF (189,190,191)
22F/22A	wchF (7,8, 192,193)	wcwA (48,49,194)	wcwV (195,196,197)	whaB (198,199,200)
23F	wchF (144,156,145,193,201)	wchV (202,203,204)	wchW (205,206,207)
33F/33A/37	wciB (208,209,210)	wciC (211,212,213)	wciD (214,215,216)	wciE (217,218,219)	wciF (220,221,222)

Explanatory notes: aProbes containing 60-bp oligonucleotides were designed and named as 1, 2, 3 etc from Group 1. The name of each GT gene (wchB etc) was derived from the Sanger Institute.

Table 3

Oligonucleotide probes used in this study.

Spot identifier	Targeted GT gene	Specificity	Probe sequence (5′–3′)
1	wchB	Serotype1	ATAAGATTATTGAGAAAATATAGACCGGATGTAGTCTTGACATATACCGTGAAACCAAAT
2	wchB	Serotype1	TTTATTGGTAGGATATTAAAAGAAAAAGGTATAGATACTTATCTGGCTGCTGCCCAAATT
3	wchB	Serotype1	GAAAATGAAAAACGAAAAGAGATGGGACTTCAAGGGAGAATGTATATAGAGCAATATTTT
4	wchD	Serotype1	TTATTGAAGGAATGATTGATAGCGACTTAATAGTTGTTCGTATTCCGTCTATAATTGGAT
5	wchD	Serotype1	GCCATAGATTTGTATTGGAAGCAATGAAGAGATTAGAAATACAAGGTATTTTGTTGGATT
6	wchD	Serotype1	AGCGATTGCGGGATCTATTATAGATTTTATTAGTATGGATAAGGAAAAGATGGTGATAAA
7	wchF	Serotype2, 21, 22F, 22A,23B, 32F, 32A	TTTGTTGAGAAATTAACAGAATATCAAAAAGATGGTAACATCCAATACTATGTTGCCTGC
8	wchF	Serotype2, 21, 22F, 22A, 27, 32F, 32A	CTAAAAAAGACTTTGTTCTCATTACAAATGTGGAACAGAATAAGTTTTACGATCAGTTGC
9	wchF	Serotype2	TTATTGAAGCAGTGGAGCAATTTGATGAGAACGCCATTTCTGAACTAGATAAAAAATCTA
10	wchG	Serotype2	GCAATACCAAGAAAAATACCCTAAAAAAATTAAGGTTATCACAGATTCCTCTGTTATAGG
11	wchG	Serotype2	TAGAAGTTTAAACAATCTGTTAGATTTGAATAGTAATGCAGTAGCTATGCATGATTGGTG
12	wchG	Serotype2	TTATCAGAATTCTCTAAGTAATGAGGAGACAGATATTATTCGTGAATTTATCAGCATTCC
13	wchH	Serotype2	TAGAAACCAGACAATTTTTTATCGGATAAAAGCTTCTTTGGGGAATACTCTAAAAAACG
14	wchH	Serotype2	CGTATTCCAGAAAAGTTACCTGATACCTATAATGTGTTGATTAATCCTGAAAGAGAAAAA
15	wchH	Serotype2	CTTTGTTGGAACTCTCAAATGGTCAGAATACTATAGTTGTAGAAGAGTTATCAGAAATAT
16	wchI	Serotype2	CATTTTACCAGAACATGGAAATGTGGAAGATGAGCTTGTAAACAAAGGAATTAAATACTA
17	wchI	Serotype2	TGATTTAGTTAGAGCGATAGCTAATCTTCCTGAGAGATATAAACAGATGTTTAAAGTTGA
18	wchI	Serotype2	TACAAAAGAGATAATTTCTACAGGAGAAACAGGATATCTGTATGAACCAGGAGATTATAT
19	wchE	Serotype3	TATAAGTCCTACAGTTGTAGTGTAAGTGATGAGAAGTTATTTAGTTCTGTAATTATCCCT
20	wchE	Serotype3	ACTTTAAAAAAAGGCTATAAAACTGTTATGCAGGATACTTCTGTTGTGTATACAGATGCT
21	wchE	Serotype3	ACTGCAATTGTTTATACAGCTTCATGGTGGGAAATTATTTTATATGTTCTTTTGGGAATG
22	wciJ	Serotype4, 45	GATTGTTGAATTATTTTAGCTTTGCAATTAGTTCTACTTTAGGAGTTTTATTGGGGAGGT
23	wciJ	Serotype4, 5, 45	GCCACAATATGCAGAAGATCTTTTTATCCCTGATGAATCTATAGTTAATAAAGAAAGTGT
24	wciJ	Serotype4, 5, 45	CCTTCTATAAAAAATCAGATGCTATGTTAGTTTCTTTAATAGGAGACTCGATAGTTTCTC
25	wciK	Serotype4	GGTTCAGAAACAATTGGTGAAAAATTCTTTAATGAATATCGTTTCTTCAGACGGCTATAA
26	wciK	Serotype4	TCGATTTCAGTTGAATTTTATAGGTACTAATGCAGGAGAATTAAGGGAATTTTGTCAAGA
27	wciK	Serotype4	GTGAAGATACTTATATGGAAAAAGTGTCAATAGAGAATGGTTTTGGTTTCGTTTTACCTA
28	wciL	Serotype4	AAAAGCCTCTACATCAGTTTCTCTCTCTTGCTAGAATAATAAAGAAAGGAGATTATGATA
29	wciL	Serotype4	AGAACTCATTTTAATCAAACCAAATGTTATTTTACTCCTAGTTGGTAATGGTGAGGATGA
30	wciL	Serotype4	AAAAACATTAGTTACTTACCTATCAACGAAGAGTCTGTGTTGCTATGGAAAGATAAAGTA
31	wciJ	Serotype5	TTACATAGGATATTAAATTATTTTAGTTTTGCTATCAGTTCCTCGATAGGGGTTCTACTG
32	whaC	Serotype5	TTTCTGACTCTCACAAGTATGATGGATTGGTATTACCAAAGAAAAATACAGTTCGCAATT
33	whaC	Serotype5	TATATCCCGAACCTCAACTTTTGAACCTTTTAACGAGAAATATCATATCCGTCAGATTAT
34	whaC	Serotype5	GAAGACTAAACTTCAGCGTGAATTGAAACTAGAAGAAGCACGCTATAAAGGAAATAGATT
35	whaD	Serotype5	AAGACGGCAGTACGCTATTTCTGTTGATGGTATAATAAATCATAGTAATATCTCACTTAA
36	whaD	Serotype5	GTTTTCACAAGATATAGTATTCGAAAATCTGAGAAAAATCTGCTTTTTGTGGGACAGTTT
37	whaD	Serotype5	TCTAATATACATATAATTCCTTTTCTAGAAAAAACTGATATCCTAGAGTTGATGCGGGTG
38	wciN	Serotype6A, 6B, 33D	AATAGATTATCAAAACAATTTGCGCAGAGAGAAATTAATTGGATAGAGAACGTTGAGATC
39	wciN	Serotype6A, 6B, 33D	TTACAAGGAGATTTAGGGGTTTTAAATGCAGTTTTATATAACTCATTTGGTGTACTTCCT
40	wciN	Serotype6A, 6B	GCAAGAAGGCAGTAATGTTGCACATATAGACCAATTTAAAAAATACTATGAAGGTAGTTA
41	wciP	Serotype6A, 6B	GGACACTTTTTATTAGGGATGATGGATCAAAAGATAAAACAATAGAAGTAATACAGAGGT
42	wciP	Serotype6A, 6B	CAGGTTTTAATCATGCATTGCTAGAGATGGTTCCTTCAGTTGATATTGATAAAGATTATT
43	wciP	Serotype6A, 6B	CTTACACATGCTGGGGTATATAATCAAACTCTTTATATGCTAAAAAAAGCTTCTGGAAAA
44	wchF	Serotype7F, 7A	ATACAATACTATGTTGCTTGTATGCGTGAAAATTCAGCTAAATCTGGCATCATGGATGAT
45	wchF	Serotype7F, 7A	AAAAATATATCCAAGAGGATTATAAGCAGTACCAACCAAAGACCACCTATATTGCCTATG
46	wchF	Serotype7F, 7A, 23B	TTGTTACAGGATACTGGTTTTGATAAAGATCCTAGGGTTAAATTTGTTGGGACTGTCTAT
47	wcwA	Serotype7F, 7A	AAGTGCATCTTTCCAACGTCAAAAAGAATTCTTTTCGTTGGAAAGTTATATTCGGAATTT
48	wcwA	Serotype7F, 7A, 21, 22F, 22A	GTGTGTTGGATTATCCGATTTTACGTAGAAAATACTTTAATCCTAAGGGGATTTTAGAAT
49	wcwA	Serotype7F, 7A, 21, 22F, 22A	CTCACAAATCAAACGAATTGACTATTATGAGCATACGACTGAGCTTTATAATATGTTTGA
50	wcwF	Serotype7F, 7A	AAATATGAAGTTATTCTAGTAAATGATGGCAGTACAGATGCTTCACCCAATATTTGTGAA
51	wcwF	Serotype7F, 7A	TATTTTATTGGGAATGATGCGGCTATTACCAAACAGTGGTCTGAAAAAAAAATTAGTGAT
52	wcwF	Serotype7F, 7A	ATGAAATTGTATGAAGAAAATCAGGAAGACACTCAACTTTTTAGGTTGATACTTGCAGAA
53	wcwG	Serotype7F, 7A	AAAACGATTACCCGGATTTTTATTCCATAATTGGTGGTTAGAAGAATGGTCTAGAAAATT
54	wcwG	Serotype7F, 7A	GGTGCAGATAAAGGAAGATTGCCAAAATTAAAAAGCTTAGCTAAGCAGATAGTTTTAAAT
55	wcwG	Serotype7F, 7A	ATAAAAAGGGACAGGATGGTCTAACCCTTAGAGCAATGGAATCCATTTTTTATAAAAAAA
56	wcwH	Serotype7F, 7A	GGAACAGAGTTACTAAGAATTGTAAAATCAAATCAATTGTAGGCAATATACGTGGCAAAA
57	wcwH	Serotype7F, 7A	ATTTGCTAAATCCTATAGAGAAACGAAACCCATTTCATCTAGGTATGTTATATCATGAAG
58	wcwH	Serotype7F, 7A	TATTTGAATATGCAATTGATGGCGAGAATGCACTTTTATCTCCGATAAAAGATAGTGTTA
59	wciO	Serotype8	AACTAATGAAGCTTGAACCGATTATGAGACAACAAGACAGCTATTTAATCACAGAATATA
60	wciO	Serotype8	AAATCACTTTATATACTGTTAAGAATACGCCCAAAAGTAGTTATCTGTACAGGTGTTCTT
61	wciO	Serotype8	TTTATATTGAATCTTTTGCAAAAGTGACCACTCCTACTTTAACAGGTAGAATACTATACC
62	wciR	Serotype8	AAAATAGATCAACTTATTGAATTAGAAGTGATAAAGGAAGAGGTGTTTGCTCAGATTGGA
63	wciR	Serotype8	TAAAGCTTTGAAATTAAGAAAAAAAATTATAGCGGTTCCACGATTAGAGCAGTTTGGAGA
64	wciR	Serotype8	ATGCTTTGATATAGAGCAGTTAGGAACTGTTTATCAAAAAGCTCAGACTTTTACAACAAA
65	wciS	Serotype8	TTTATTGATGGCTCTCTTGTAACACGGTTAACCTATTCTAGTTATGCTCTTCTTAAATTT
66	wciS	Serotype8	AACAACTTTCTTTTTTTAGGAAGGATGGGCAAAAGAAAAGGAGCCTATGATTTAATAGAT
67	wciS	Serotype8	AGGATAATGGCTGGTTAATTCAACCGGGTGATATTTCTCAGTTATCTAATATTATTTTAG
68	wciT	Serotype8	ATGGAATGAGGATAATTTTGATTTATCAGATTCACAATTTGCGAAGTCTGCATATGAATC
69	wciT	Serotype8	GGTGCAATATTATGAGCAAGCAAGTTTTGATATCAATCATTTGGTAACTGTCAATACAAT
70	wciT	Serotype8	AATAATTGATGGATTAGCAATTTATCCAGATGATTACTTTTGTGGTTATGATCAGGAGGT
71	wchO	Serotype9A, 9V, 9L, 9N, 19A	ATTAACGATGAAAGAAACAGTGGATGCTGTTGAACAGTATGTTTTAAAGAAGCATCCTTT
72	wchO	Serotype9A, 9V, 9L, 9N, 19F, 19B, 19C	TTTTGATGTATTATCAGGACACATTAAACGAGCTCCATTATGGATGCAAAAATTGAATCT
73	wchO	Serotype9A, 9V, 9L, 9N, 19A	GAAAGAATATATTATCCAATCATTCATGGATAATGGAATTAATGCTGTGTTTATGGGGGT
74	wchO	Serotype9A, 9V, 9L, 9N, 19A	GAGTAGCGGGTATTGATTTGATGCAATGTCTTTTAGAGTTGTCAAATAAAAAAGGATATT
75	wcjA	Serotype9A, 9V, 9L, 9N	AACAGGTGGACTATGGGAGAGCAAACTTTTATCAAAAGGAGTTCAACATCATAAAATTTT
76	wcjA	Serotype9A, 9V, 9L, 9N	TTAAAAAAAGCGTATTGTGTAGCTGTGGGTAAAGCGGTTAATGATAATTTGAAACATGAT
77	wcjA	Serotype9A, 9V, 9L, 9N	GTTGTTGAATGTATCAATAGTTTTGATTACTTAGTGTCATCATCTTTATATGAGGGGTTG
78	wcjB	Serotype9A, 9V, 9L, 9N	GAAAAGGCTAATTTAGAAAATGAACTAATTGTTTCGTTTACAACAATTCCAAGCCGTCTT
79	wcjB	Serotype9L, 9N	AGTTGTTCTAGTTGATGACGATATCATTTATCCTCGAAATACTATAAAGAAACTGATTGC
80	wcjB	Serotype9L, 9N	CAATCCTGAGGAGAGTTTGGTATATTTGAATACCGTATATGATAACAACAATGATAAATG
81	wcjC	Serotype9A, 9V, 9L, 9N	AAATTTCTAGCTGAACAACTTGTAAAAGAAGGACATGAGGTATTTGCATACTCTGATGAT
82	wcjC	Serotype9A, 9V, 9L, 9N	TTATCAATAAAGGATTTATTAACCCATCTTCTCAAAAATGTATGGCCATTGAAAATGCTG
83	wcjC	Serotype9A, 9V, 9L, 9N	CGAAAGTGATCCTAGAATACAATATTTAGGCTTTCAAGATACAAAAAACCTCTATGAAAC
84	wcjB	Serotype9A, 9V	ATTGTAATTTTGGTTGATGATGACACTGTCTATTCATCGAATACCATCGAAAAGTTAGTT
85	wcjB	Serotype9A, 9V	ACCCTGAAGAGAGTTTGGTGTATCTGAATGCTATATATGATAATAATAATGATAGGTGTA
86	wciB	Serotype10F, 10A, 10B, 10C, 47A	ATCAAGGTAATCATATCTCACACCTCAATCCTTATTATTGTGAATTGACAGGATTATACT
87	wciB	Serotype10F, 10A, 10B, 10C, 31, 47A	TTTAGATGTAACGCGAGAAATTATAAAAGAGGTTTCGCCAGAATATTTAGCAACATTTGA
88	wciB	Serotype10F, 10A, 10B, 10C, 47A	TGAATTTATTTGAGAAGGGCAAATCCTTCTTGAAAGCCAAGTATTTCGGAAAAAAATATG
89	wcrC	Serotype10A, 10C, 34, 35F, 43, 47F, 47A	GTTGCTGTATCTTTGGCAAACGAACTTACAAAAAAGTATGAAGTTCATTTGATTGGAATT
90	wcrC	Serotype10A, 10C	TAACTGTTGGTCGTTTTGATTATCAAAAAGGATATGATTATCTTATCCAAGTCGCGAAAA
91	wcrC	Serotype10A, 10C	TGGTTATCTGATAGATTGTTATGATACCGATAAGATGAGTGAGAAATTGCTTGAATTGAT
92	wcrD	Serotype10A, 10B	GGATATGGTTCTTACGGATTTTACAGAACAACATGTTTATAACAATACTACTGTTCGAAA
93	wcrD	Serotype10A, 10B	ACCTATAGAACATCTATCCTAGTCGACAATAGAATTCGTTTAAGTGAAAAGACGTTTTAT
94	wcrD	Serotype10A, 10B	TATCAGAAGAATTATACAGACAAATTGAGCAGAGTTCTTATGAGTATATCCCTACGAAAA
95	wciF	Serotype10A, 10B	AAGCATCATCAGATTGGATTTTCTTTCTAGATCCAGATGATTATTTGGAAGATTATACTC
96	wciF	Serotype10A, 10B	GGATAAAATTGTGATTAGTCCACTTGAAACATATAACTATTACCGTAGAGAAGGTAGTAT
97	wciF	Serotype10A, 10B	AGGCTGACTCTGGTTTAACAGATTTTTCGAAAGATCGAAACCTATTAAAAGTTGATTTTA
98	wcrG	Serotype10A, 10B	CTCTGTTGGATTATAAGGAACATGATATTTTTATTATTGTAGGCAGCAAAGTTAATGTGG
99	wcrG	Serotype10A, 10B	GCTAGAAATATTCAGAACAAATATGTTCGTAAATTTGTAGCATATTACCGTAAGCTAGAG
100	wcrG	Serotype10A, 10B	GCATCTAACTGGGTATCTATTAATCAGGATTTAGTTAGAATAATACTAGAAGAAGAGAAA
101	wchK	Serotype11A, 11B, 11C, 11D, 14, 15F, 15A	GATAGATTAAAAGGTGAGGGATTTATTCAGGATGATGTTTTTATTCAGACTGGTTTTTCA
102	wchK	Serotype11A, 11D	TTCTTATGATGAGATGAATCGCTATATAGATGAAGCAAATATTATCATTACACATGGCGG
103	wchK	Serotype11F, 11A, 11D	CGAAGGGTATGAATTATCTCTGATTAATGATATAAGCGAATTGCAGTATAGTTTAAAGCA
104	wcyK	Serotype11F, 11A, 11D	CATCATAAATTAGATAAACTACTACGACCGATATTTTATCGAGTTTATACTCAGGCATGT
105	wcyK	Serotype11F, 11A, 11D	CTAGGTCATGTTGGACGTTTTAATACTCAAAAAAATCAATGTTTTCTAGTGTCTCTAATG
106	wcyK	Serotype11F, 11A, 11D	GGTCAATTTGATGATATGAAATCTTTTGTGTCATCAATGGATATAATGTTGCTTCCAAGT
107	wcrL	Serotype11F, 11A, 11D	TGTATTGAAAATCAAAATCAATTTGTGCAGGATGCATATAGAGATAAAGCATGGGCTTTT
108	wcrL	Serotype11F, 11A, 11D	GTTATGTCCTGAATTAAATACACCTGTATTTAAACGTCTTGGTTATACTTATTCTGACTG
109	wcrL	Serotype11F, 11A, 11D	CAGATAAAACATTCTCTATTCATCATTATAGTGCTTCTTGGACTTCCTTAAGAAATCAGA
110	wciJ	Serotype12F, 12A, 12B, 44, 46	GGAAATATATGCTGATTATCGTAAGAGAAAAAAAAGAAGAGAGACTATAAATGGTGTTGC
111	wciJ	Serotype12F, 12A, 12B, 44, 46	ACTTAACTTTTGCTGGAAATATTGGAAAAGCTCAGAATTTAGAGACTATTTTGAAAGCAG
112	wciJ	Serotype12F, 12A, 12B, 44, 46	AATGTTGATCAGTTAGTGAGAAATATTCGTAAGTTCTGTTTGCTTTCTGTAGAGGAAAGA
113	wcxB	Serotype12F, 12A, 12B, 44, 46	ATGTTCCGAAACAATTTCAACAGTATGCAGTGAAAATTGGTACAAAGTCTGATATTCGTT
114	wcxB	Serotype12F, 12A, 12B, 44, 46	AAAAGAATATCCAGTGAAAGTAATTCATAATGGTATTGATACTACTGTCTTTCAACCGAG
115	wcxB	Serotype12F, 12A, 12B, 44, 46	TAGAAAGTGCTAAACTTTATGGTCTCGTTTGTCAGGATAGAAACGTAGCTTCTATTTTAT
116	wcxD	Serotype12F, 12A, 12B, 44, 46	GAAGAAGAATTTTTTTAAAGTTAGTGGAGCTTTGCGAAAAGTGTTGAAAAAACAGCAGTT
117	wcxD	Serotype12F, 12A, 12B, 44, 46	TGTCAGCTCTCTTCTAAAAAAATTATCAGTGTTGGATCTTTAGTACGACAAAAAGGTTTT
118	wcxD	Serotype12F, 12A, 12B, 44, 46	GATAGAGAAAAATTAGAGGAGAAAGTCAGGGAATACCAATTAGAAGGCTTTATAAATTTG
119	wcxE	Serotype12F, 12A, 12B, 44, 46	ATAAAATCCCCGATAATCTTACCCAATTTTTTGGACGAGAAAATATAGAAGAGAGAGATA
120	wcxE	Serotype12F, 12A, 12B, 44, 46	TATAAAACCTTGATTACTCCCATTTTGATAAAAGAACAGATACCAATTATTCGGACGCAA
121	wcxE	Serotype12F, 12A, 12B, 44, 46	AGGTAGCAGATTTTGCTTTATTTCCTAAACAATGTAGTTTAAGTTTTTATGATGCACAGG
122	wcxF	Serotype12F, 12A, 12B, 44, 46	AAGTTACAATGAGAAATATAATCATGATGAAATTACGGTCGTTAGTTGTGACCATAAGGA
123	wcxF	Serotype12F, 12A, 12B, 44, 46	TGATTGTTTTTTTTGGACGTATCAACAAAAATAAAGGTATCAAAGAACTGCTTGAAGCCT
124	wcxF	Serotype12F, 12A, 12B, 44, 46	GAAATGCTCTTCGGTTATTACTTCTAATAGAGATAGAGGAGCCTATTTTTCTATTGAAAA
125	wchK	Serotype13, 14, 15B, 15C	CTTATGAAAAAATGAATCAATTGATTAAGGAATCAGATATTATCATTACCCATGGCGGTC
126	wchK	Serotype14	TAAAAATCCAATAATTGTTCCGCGGCTAAAAAAATTTGGTGAGCATGTAAATGATCACCA
127	wchK	Serotype14	AGGACAAACATTTTGAAACTTATTTGAATAACGAGAGATTTAATGTACGTTTCAATGTGG
128	wchL	Serotype14, 15B, 15C	TTGTGTTGATAGTGCCTTAAAGCAAAATTTAGAATCTCTTGAAGTGATTTTGGTGAATGA
129	wchL	Serotype14, 15F, 15A	AAAAATTCTTGAACAGTATGGTGATAATCCCCAAGTGATGATTTTCCATCAAGTGAACAT
130	wchL	Serotype14	GCTAAGTTATTTCTTCGTAGAAGAATTGAGGAAAACAATATTGCTTTTTCGACTGAAATG
131	wchL	Serotype14, 15F, 15A, 15B, 15C	TCCTAAAATTGAGGAGAACTACTACAAGCAACATATGGATTTTAGATTTTATCTTGCTAG
132	wchM	Serotype14	AATAGAAAGTATTTTGAATCAAACGTATGATAACCTTGAGGTTCTATTAGTCGATGATGG
133	wchM	Serotype14	AATAGAAAGTATTTTGAATCAAACGTATGATAACCTTGAGGTTCTATTAGTCGATGATGG
134	wchM	Serotype14	CAGTATTGTAACTGGATTGTTACAATAACTGTTAGTCATTACAATGTTTTGAATGTAGCC
135	wchN	Serotype14, 15F, 15A, 15B, 15C	CAAAAAAATGATATGAACATTTCGAATAAAGTTTGGATTTGTTGGTTTCAGGGCGAAGAA
136	wchN	Serotype14	TATGCGAGAAAACTACTCTGGGAGTATTGGCGTAGAAAAAATAGTTTATGCAATTATTTT
137	wchN	Serotype14	GAGTTAAATAATCAATTTTCAGAAAAAAGGTGGGAACAGCTAAAACAGATATCGGTGTTT
138	wchK	Serotype13, 15B, 15C	GATGAAGTATTTATTCAAATAGGATATTCCAGTTATATTCCGAAATATTGTGAGTGGGAA
139	wchK	Serotype15B, 15C	GCATGTGAATGACCATCAGCTTCAATTCGTAAAACTGACGAAAGAAATATACAATTTTAT
140	wchL	Serotype15B, 15C	AGAAATTTTGAACCAGTACGACAGGAATTCAAGGGTTAAGATTTTTCATCAGCTTAATAA
141	wchL	Serotype15F, 15A, 15B, 15C	GAAGAAAATAATATTACTTTTTCGACTGAGATGTCACTAGGTGAAGATATGTCATTTGTG
142	wchM	Serotype15F, 15A, 15B, 15C	GAAAGTATTTTGAATCAGACTTATCAAAATATCGAGATTTTATTGGTTGATGACGGAAGC
143	wchM	Serotype15F, 15A, 15B, 15C	GTACTGCAATTGGATTGTTACAGCGACTACCAATCATAGTAAGATTTTAAATCCTAATTT
144	wchF	Serotype7B, 16F, 17F, 18F, 18A, 18B, 18C, 23F, 23A, 24F, 24A, 24B, 28F, 28A, 40, 48	GAAACTTTTGTTGAAAAATTAACAGCCTTCCAACAAGATAAGGCTATCCAATATTATGTG
145	wchF	Serotype16F, 17F, 18F, 18A, 18B, 18C, 23F, 24F, 24A, 24B, 28F, 28A, 48	AAGGTCTTATGGTCAAACATGCAGCTCTTTTAGTGTGTGATAGTAAGAATATTGAAAAAT
146	wchF	Serotype16F, 17F, 18F, 18B, 18C, 23A	TTCGTTACTTGAAGCATTAGCATCCACAAAGTTAAACTTACTACTCGATGTTGGTTTTAA
147	abp1	Serotype17F, 24F, 24A, 24B, 48	GCCAGTCATTATCTATACCCTTGAAAAATTTCAAAATCATCCAGAAATTGATGAAATCTG
148	abp1	Serotype17F, 24F, 24A, 24B, 48	ACACAAACTCCTCATGTTTACCATCTTGATAATATTCTATCGCTTCATGAAAAAGCATTA
149	abp1	Serotype17F, 24F, 24A, 24B, 48	TTATTTCTCTCTTGGAACAGAGAAAAACTTGAAAATTACGACTGTAGAAGATCTCGATAT
150	wciP	Serotype17F	GAAGAAAAAGATAGACGGATTAAATTGATTGAAAACATATCGGAATATCATGGAGCCTAT
151	wciP	Serotype17F	GTATACCAATCCTATCTCAACTTTTATGGCTCATAAGGTTTATGGATGTAATACGTTATT
152	wciP	Serotype17F	ATCTTAAAACGTATCTCGAAAATTGATGAATTAGCTAAAGATCATGCCTTGACTTACAAG
153	wcrV	Serotype17F	TCGACAGATAGTAGCAAACAGATAATTAACGAGTATCTTAATGCAGACAGTAGATTTAAA
154	wcrV	Serotype17F	CATGCAAAACTTAAGTTGTTCTGTCAGAATTTTAAGTTAGTGAGGAAACAGATTTTTAGG
155	wcrV	Serotype17F	CGATTTAATCTACTAAAAAATAACGGAGGAATGTGGGTTGACTCCACTATATATTTTACT
156	wchF	Serotype7B, 16F, 17F, 18F, 18A, 18B, 18C, 23F, 23A, 24F, 24A, 24B, 28F, 28A, 40, 48	TATAGCGTATGATATCGCTGCAATTAACAGAGCTATTGAAATTGCCAAAGAAAATAAGGA
157	wchF	Serotype16F, 18F, 18B, 18C	TATAATCAGCTATTAGCAAGTACTGGATTTGATAAAGATCCACGAGTGAAATTTGTTGGA
158	wciU	Serotype16F, 18F, 18A, 18B, 18C, 28F, 28A	AGAAAAAGTACAACCCGACATTATACATATTCACTCGTTTATGGGATTGCATAAAGAATT
159	wciU	Serotype16F, 18F, 18B, 18C, 28F, 28A	TCATCATCAGAGATTGACAACTGCAAATAATAAAATTAGAGTTGCTTATATTGGTCCAGA
160	wciU	Serotype18A, 18B, 18C	GACAAGGAAGATTTGTTGGCTAAAATCATCAATAATCAGTTGAAGAAAATTCCGCTTAAA
161	wciV	Serotype18A, 18B, 18C	AAATACATAACCTTTGTAGATTCAGATGACTATGTTTCTCTAGATATGCTGCAAACTCTA
162	wciV	Serotype18F, 18A, 18B, 18C	AGAAGATGCTATTTTTCAAATTGATTGTTTAAAATTAGCAACATCTGCCCTTGTTATCCC
163	wciV	Serotype18F, 18B, 18C	ACCCAATATCAAAATCAGTATTACGTCATTATCCAATCCATCGTTTACCTTTTACTAAAC
164	wciW	Serotype18F, 18A, 18B, 18C	AAGTGCAACTTGAAGATAGGGCCTACAGAATACTAAAAAAGAAATACGGTTCTTTAATTT
165	wciW	Serotype18F, 18A, 18B, 18C	TGGATTGACTCAACAGTGTATTGTACAGGAATTACTACCATAGAGACAATTGAAAAAAAT
166	wciW	Serotype18F, 18A, 18B, 18C	TACGAACGCAACACCACATATAATGGTTGATGAATTAAATAATGTTTTTTCAAAGGAACG
167	wchO	Serotype19F, 19B, 19C	ATAGATAGTGTAGAACAATATGTATTAGAAAAAAGACCACTACACTTGATGGGGGTGAAT
168	wchO	Serotype19F, 19B, 19C	GCTCAAAGTATTTAAGAGAGATTATCCAAATTTGATAGTTATTGGACACAGAAATGGCTA
169	wchO	Serotype19F, 19A, 19B, 19C	AATTTAGAGTGGTTATTCCGTGTAGCTAATGAGCCTAAACGTCTCTTTAAACGTTATTTT
170	wchO	Serotype19A	GAGTTGCTGGAATAGACTTGATGAAACATTTACTAGAGTTGTCTAATGAAAAAGGATACT
171	wchQ	Serotype19F, 19A, 19B, 19C	ATCAGATTTAGAAATTGATGTTTTGATTAACCATGAAAATGCTGGTTTTGCTCGTGGAAA
172	wchQ	Serotype19F, 19A, 19B, 19C	ATCAGTAGACTATAGAAAACAGGTAGAAAACCCAATTCTTCATGGTTCTTTTATTGTATA
173	wchQ	Serotype19F, 19A	GGATACAAGAGAATTTATACACCTAAAATTAGAGTTTTGCACCATCAAAATGTTGCAACT
174	wciB	Serotype20	ATACTGGGGAAAACATTTCCCAGTTAAACCCTTATTACTGTGAATTAACAGGTTTATATT
175	wciB	Serotype20	AAAAAGGAAATATTATATTGAAACTCTATGTTCTCATTATGCACACACGCTAGATGCTAG
176	wciB	Serotype20	AATGGCTGTTTCCGATTTTAGATTGTATGTTTGATCAGATTAATCTTTCAGAGTTAACTG
177	whaJ	Serotype20	TTTCTCAAAAATTAGCGACCGAAAACTCAAATATACGAGTCTTGAAATCAGATAAAGGAA
178	whaJ	Serotype20	GATTGATGAGTACGGTTTGAAGTTTAATACGAATTTGAGAGTTTCAGAAGATAGTGATTT
179	whaJ	Serotype20	CTATGTTTTTTGAGCCTATACAAAATCTATCTGTATCTAGTGTTAGCAATTTATCGCTAG
180	wciL	Serotype20	GATACGTTATTATTGGGAAATGTATAGATTCTTCAAAGAATATGCATCTGATTATCAGGC
181	wciL	Serotype20	TATACATTAGACAATAAATTTGTGCTAGGTCATGTAGGACGTTTGCATTTTCAGAAGAAT
182	wciL	Serotype20	GACACTACTCTCAGAAGAAGGTGTACCAAAGGAAGTAAAAATCAATGATAATACTTTTTT
183	wcwK	Serotype20	AAACAAGATATAGAGATATGGATTTGTTTCAATATTGGTTTCGAGCGGTAGAAAAACATG
184	wcwK	Serotype20	AATCTATTTAGCATTTTTTATTCAGGGATTATTGGTTATCATGATGCTCATGTCGCTATG
185	wcwK	Serotype20	GTGAATATGTGCCTCTGGCTTATTCAGGTAAAATTGAATCTATTATTCACAAACAAAAGA
186	wciD	Serotype20	TGGCTCAGAAACTGGAAAAAGAGTATTCTGGCATAGTTAGTATAATTGATAAAGAAAATG
187	wciD	Serotype20	CATAAAAATTGATGAGAATATGTTCTACGTTGACATGGAGTATATTGTTTTTCCAACTCC
188	wciD	Serotype20	GAGACAATTGCTAGATGTGTTACTATTATGACAAATGTTTGTCTATCAATGGAAGATACT
189	whaF	Serotype20	ACTTTAATACAAAAAACTGAATTTCCTAAATTTATCTGGACTATGTGGTGGCAAGGAGAA
190	whaF	Serotype20	ATTTGGTTAGATTCAACGATGTATGTCCATCCAGATTTCCCTATTGAAATATTAGAAAGA
191	whaF	Serotype20	AGGGAAATAATAAAAAGTATCCCTAGATATTCTAGTCAAGAAGACATCTTTTGGTTGAGA
192	wchF	Serotype22F, 22A	ACTTATATCGCCTATGGAACAGATACAAGCAAGTCTATTTTAAAACCTGATGACGAAAAA
193	wchF	Serotype18F, 22F, 22A, 23F	ATCGCTTTTAGAAGCTCTTGCTTCAACAAAGCTTAATTTACTGCTAGATGTTGGCTTTAA
194	wcwA	Serotype22F, 22A	TAAGAAGACAAGGAGAATCGTTTTCTTTGGAATCTTATATCCGTAGTTTCTCAGAATTAT
195	wcwV	Serotype22F, 22A	GAAAAACGGGGAAAAAATTAAAGTATTTTGGAGAAGGGGAATAAGATTATTTAGAAGTGG
196	wcwV	Serotype22F, 22A	GGAGAATAAGCAAAATATTCTTTATGTAGGCTCACTATCAAAAAGAAAAAACACAGCTCA
197	wcwV	Serotype22F, 22A	ACCTTATTTAAAGAACTCTCAGCTTCAATTTATTTACCCATCATCACAACTATTTGTGCT
198	whaB	Serotype22F, 22A	TGGCAGTATAGAAAGGGTAGAAGCCTTATTTGCAAATAATGACGAGATAGTTATAATAAA
199	whaB	Serotype22F, 22A	CATCATCAAAGTCCTGTTGTTGAGAAGATCAATTCTATATCTAAGGCAAATAAAGAACTT
200	whaB	Serotype22F, 22A	TTATTTTACATGGGAGTTGTGTAATTTTTTCACCATTATATGTTTCAGAGGAGGAGTTTG
201	wchF	Serotype23F, 23A	CCATTTACTGGAAGAAAGATAATCTTCATGAGATTATTGAAACGAGTGAACAAAAAACAC
202	wchV	Serotype23F, 23A, 23B	CCTCATTTTTGTTGACAGTGATGATTTTGTCTCTCAAGATATGGTATCTTATTTAGTATC
203	wchV	Serotype23F, 23A	GGCCAAGATATTTAAAAGAGAGTTGTTTGATGATATAAGATTTCCTGTAGGTAAGCTATT
204	wchV	Serotype23F, 23A	TTTTGGAGATTACGAACACAATTATTAATCACTATGGTGATAATTTACGCGTGTATACTG
205	wchW	Serotype23F, 23A	ATTTGAAACAAAATTATCAAATAAACTTGGCCTACAAAAATCTTTGCATGGAAAGGGTGG
206	wchW	Serotype23F, 23A	CGGGGGGATATTATACAAAAGAGTATAAACAACTATTCAGTTCGGTAGTAGAAAATATTA
207	wchW	Serotype23F, 23A	CCTATAGAGTAAATCTCCATCAATTTTTAATAAACGAGATCTCAGATGCTACAGTAAGAT
208	wciB	Serotype33F, 33A, 34, 35A, 35B, 35C, 37, 41F, 41A, 42	TTGGTTTTATCGGTGATAATACTGGCGATAATATATCCTCTCTAAATCCATATTATTGTG
209	wciB	Serotype33F, 33A, 34, 35A, 35B, 35C, 37, 41F	ATAGTTCCAAAGAAGCGAAAGTATTATATTGAAACTCTTTATTCACATTATGCCCATACC
210	wciB	Serotype33F, 33A, 34, 35A, 35B, 35C, 37, 41F	AACTATTAGATGATTATTTACCGTGGCTTTTTTCTATTCTGGATACTATGTACGAACAGA
211	wciC	Serotype33F, 33A, 37	CAAATTTTAATATCTGATACAGATGTTTATTATTTTACTCCAGCTGGTTCAGTAGCTGGT
212	wciC	Serotype33F, 33A, 37	TTACGAAATTTTATTAGAAGTTGCTAAGAAGATGGTGGGGGATGAGAAATATCACTTTTA
213	wciC	Serotype33F, 33A, 37	GTTTTACCATCGTATTATAAAGATGAAACTTTACCTATCAGTATGTTAGAAGCAATGGCA
214	wciD	Serotype33F, 33A, 37	AATAGCAAGACAATTCGAGAGAGAATATGAGGGAATTGTTAGAGTTATAAGTAAGGAAAA
215	wciD	Serotype33F, 33A, 37	TGCAAGAGAACAATATTCGGCTGTCTGAAAAAATGTTCTATGTAGATATGGAATATATTG
216	wciD	Serotype33F, 33A, 37	GCATAAACAAGTGATCTATCATTTGGTTGATTTTTATAATCAAATGAGATCTAGCGCTGT
217	wciE	Serotype33F, 33A, 37	GCCAATTTTTAAAATCCTATAACTTTAAAGAGGTATCGCACAAGGAGATAGAACAAAGAA
218	wciE	Serotype33F, 33A, 37	CTGAATTATTTAAAAAAAGATTTTTATACTATTCGAGCAAAGACACATGAGAGAGTGCCC
219	wciE	Serotype33F, 33A, 37	TAAAGTCGAAGAAAATAATCAGGAGTTGTTCTTTTTGGCAGACAATTTTTCTAACCAGTA
220	wciF	Serotype33F, 33A, 37	CTGGGGAAATATGTGATGAATATGGGAAACTGTATGATAATATTCATGTTTTCCATAAGA
221	wciF	Serotype33F, 33A, 37	CAGAGCGTTTGTTGAATATTAAAACAGTTGCTCATACCGATTTGCCTATATATCATTATT
222	wciF	Serotype33F, 33A, 37	AAGGAATTGTTAGCAGCCTTAAATGCTAAAAGAGTAATTGGCTCCTTTATTTTGAGTAAT
223	16S	Streptococcus pneumoniae	TATTGGAAACGATAGCTAATACCGCATAAGAGTAGATGTTGCATGACATTTGCTTAAAAG
224	16S	Streptococcus pneumoniae	ATAAGTCTGAAGTTAAAGGCTGTGGCTTAACCATAGTAGGCTTTGGAAACTGTTTAACTT
225	aroE	Streptococcus pneumoniae	ATTTCAAAAACGGTGTTTCAAAGGGTTGGTATATGATATCTGCAACTAAGAGAGTTTCTG
226	aroE	Streptococcus pneumoniae	AGCAGGGTCATCTTTTTACCTGAAATTGTAAAAGAAGGCAAGCACTTAAAAAATCCCTTG
227	aroE	Streptococcus pneumoniae	TCAAAGGCTCTATTGTGGATGAAGGGAGAAATAGAATGCTTAATAGGATTGGCAACAACT
228	ddl	Streptococcus pneumoniae	TATTGAGCTCGTTGAGAAAAATCTCTCCCTTATCTGTATAGAAGAAATCGCAACGAGATA
229	ddl	Streptococcus pneumoniae	ATGTTTGACGGCTTAGTGAAGACTGGATAAGCCAATTTTTCTTCCACTTCAGCGATTTTA
230	ddl	Streptococcus pneumoniae	CATGACTAAATTCCTGTGTTTTGATAAAGTCACCTGACTGACTGATAAAGAAAGTCTTGA
231	gdhA	Streptococcus pneumoniae	TGAATTCCTCCAAGCTGTTGAAGAATTTTTCAACACTTTGGAACCTGTATTTGAAAAACA
232	gdhA	Streptococcus pneumoniae	TAAACCAAGGGATTTTGAAATTCCTCGGATTTGAACAAATCTTTAAAAACGTCTTGACTG
233	gdhA	Streptococcus pneumoniae	TATACTGAAGAAATGCTCAAAGCTAACGGTAACAGCTTTGCTGGTAAGAAAGTGGTTATT
234	gdhA	Streptococcus pneumoniae	ATGGACGTCTCAAAGACATCATGACCAACATCTTTAACACAGCTAAAACAACTTCAGAAA
235	glcK	Streptococcus pneumoniae	AAATGGTCAATCAAGACCAACATTTTGGATGAGGGAAGTCATATCGTTGATGATATGATT
236	glcK	Streptococcus pneumoniae	AACAAAAGATTGAAAAAGCTTTGGGCATTCCATTTTTCATCGATAATGATGCCAACGTAG
237	glcK	Streptococcus pneumoniae	AGAATTCCTTCTACAAGGTGTTCAAAAAGTTTACGATGAAAATAGTTTCCCACAAGTACG
238	spi	Streptococcus pneumoniae	AGAAGGTATTCTCCTTCTGGAACAGTAAAGCTAAAGTTGGTGTTGTAGTTGACATCAACT
239	spi	Streptococcus pneumoniae	TGATATAGTCTGCTAGATAAGGCTCGTCCGTTTCTTTGTCATTGATGTAGAGTTTATCAT
240	spi	Streptococcus pneumoniae	AACATTGCTCCAAAAAAAGATACGGCTCAAAGCTAGTAATGACAGAATCAGGAGGAATAA
241	tktA	Streptococcus pneumoniae	ACAATTTTTACAAGATTTTCTACAGTAAAGCCATATTCTGCCAATACTTTTGGTGCTGGG
242	tktA	Streptococcus pneumoniae	CAATCAAGATGGTATCAAAGTCGGCTGCATTTTCATATACAACATAAGCACCTTTAGCAA
243	tktA	Streptococcus pneumoniae	TTCAAGATTGTTCCCATTGCAAATTCACGAACACCAAACTGAATGTTACGATTCAAGCGA
244	tktA	Streptococcus pneumoniae	AACATGTTCTTTGAAATCAGCATATACTTGTTCTGGAATTTCAAATGGTTCGTAGTCCCA
245	tktA	Streptococcus pneumoniae	TCTCCACAGATAACGTAAGTATAGTGGTCAAAGATATTGTAGCCTTCACGGTTATATTTG
246	xpt	Streptococcus pneumoniae	AGATTCCTTTTTAACCCACCAAGTTGACTTTAGCTTGATGCGAGAGATTGGTAAGGTTTT
247	xpt	Streptococcus pneumoniae	ATGATTTTCGCCAAAAAAGCTAAGAACATCACCATGAACGAAGGCATCTTAACTGCTCAA
248	xpt	Streptococcus pneumoniae	TTTGATTATCGACGATTTCCTTGCTAATGGCCAAGCTGCTAAAGGCTTGATTCAAATCAT
249	KP_gapA	Klebsiella pneumoniae	GACGTTGTTGCTGAAGCAACCGGTATCTTCCTGACCGACGAAACCGCTCGTAAACACATC
250	KP_rpoB	Klebsiella pneumoniae	AACGGTGTGGTTACTGACGAAATTCACTACCTGTCTGCTATCGAAGAAGGCAACTACGTT
251	KP_mdh	Klebsiella pneumoniae	TGTACGATAAAAACAAACTGTTCGGCGTTACCACGCTGGACATCATCCGTTCCAATACCT
252	KP_pgi	Klebsiella pneumoniae	CCTGGCCTTTGGTAAATCCCGCGAAGTGGTTGAGCAGGAATATCGCGATCAGGGTAAAGA
253	SA_arcC	Staphylococcus aureus	TGATAGGCTATTGGTTGGAAACTGAAATCAATCGCATTTTAACTGAAATGAATAGTGATA
254	SA_aroE	Staphylococcus aureus	AAGTTTTGATTGGTCATTAGTTCCTGGTTATATTGTTGCTCAAATGTTAGGTGCAATTGT
255	SA_glpF	Staphylococcus aureus	TAAGAATTACTTTGCCAACTTTTTAAGTGAGATTATCGGAACAATGGCATTAACTTTAGG
256	SA_gmk	Staphylococcus aureus	CGTAGATTACTTTTTTAAAACTAGGGATGCGTTTGAAGCTTTAATCAAAGATGACCAATT
257	LP_acnF	Legionella pneumophila	CGAAAAAAAGGGGTTGTTGGTAAATTTGTTGAATTTTATGGTCCTGGACTTAATGATTTA
258	LP_mompS	Legionella pneumophila	TCAATGTGAACTGGTATCATTTTGATAACGACAGTGATCACTGGTTTGATTTTGCTAACT
259	CP_groES	Chlamydophila pneumoniae	TTCTTTACCTGCGTTACTTGCAATTTGCTTTAATGGAGCTGTTAATGCTTTTAGAATAAT
260	CP_gyrA	Chlamydophila pneumoniae	GTTTGGTGGCTAAAAATAAGAAGCCGGCATTATCAAAATTCTTAATATTCAATATAGCTG
261	CP_gyrB	Chlamydophila pneumoniae	CCAAGACCTTTATACCTCTGAATTTCTATGCCTTTTCTTCCAAGATTTTTAAGATAGTTA
262	CP_dnaA	Chlamydophila pneumoniae	CCTGCTGCTTCTAAAACATCTTTTAAAAGAGTTTTCACATCATCTTCATATAGTAATTGG
263	CP_accA	Chlamydophila pneumoniae	TGATAACAGTATCGATAATGCCAAATTGTTTTAAGTTTTCTCCATGCATTTTCAACATGG
264	CP_dnaK	Chlamydophila pneumoniae	TCAAAAAACAAGAAGGCATTGATCTTAGCAAAGATAATATGGCCTTACAAAGACTTAAAG
265	MP_gyrB	Mycoplasma pneumoniae	AGGAACCTTTATTTGAGGACATTATCTTTGGTGAAAAAACCGATACTGTTAAATCAGTTA
266	MP_gyrA	Mycoplasma pneumoniae	ACAAGATCAAATTGACAAAATTCGTCAGGAATTAGCACAATCAGCAATTAAAAACATCTC
267	MP_dnaJ	Mycoplasma pneumoniae	TTGCGCAAGCTCAAGGAATTTATTAAACCTAATCAAGAGGTAAAACAATATTTAAACGCA
268	MP_lgt	Mycoplasma pneumoniae	TGGGATTGCCTTTGGCATCTTAATGTTTGTCTTGAAGTTAATTTACTTTTACAAGATTCA
269	MP_fus	Mycoplasma pneumoniae	TAAGCTTCCGTGAAACCTTCAATAAAGAAAGTGAAGTTGAGGGTAAATACATTAAACAAT
270	MP_lspA	Mycoplasma pneumoniae	TTTGAAGAACTGAATTAAAAAGGAGAGGAACAGACCAATAAAACTAAAGGTAATGCAACA
271	PA_trpE	Pseudomonas aeruginosa	TCACCGAAAAAATGGTGATCGAACGTTACTCCAACGTCATGCACATCGTGTCCAACGTCA
272	PA_nuoD	Pseudomonas aeruginosa	GATCATGATGGCGGAGTTCTTCCGTATCCTGAACCACCTGCTGTACCTGGGCACCTATAT
273	SP_gki	Streptococcus pyogenes	ATTCAGCCATCAAAGCAGCTATTGACAATGGTGAAGGTGTTACCAGTAAAGACATTTTCA
274	SP_xpt	Streptococcus pyogenes	ATCGCTGGTAAATTCCTATCTAAAGAAGACAAGGTTTTGATTATTGATGACTTTTTAGCT

Figure 1

(a) Microarray oligonucleotide probes layout. Oligonucleotides 1 to 222 are provided in Tables 2 and 3. P represents S. pneumoniae housekeeping genes and 16S rDNA positive control oligonucleotides. N indicates negative control oligonucleotides designed from housekeeping genes of other bacterial species. E denotes empty spot. (b) Scanned microarray images of S. pneumoniae genomic DNA hybridized with 6 samples (serotype 3, 9V, 11A, 19F, 22F and 22A). The numbers correspond to the spot identifiers given in Tables 2 and 3, and Figure 1(a) P indicates positive spot.

3.2. Evaluation of the Microarray

A total of 274 oligonucleotide probes were used in this microarray, including positive and negative controls and GT gene-specific probes. The microarray probes were tested using 36 pneumococcal isolates from 23 vaccine-associated serotypes and 19 additional pneumococcal isolates belonging to other serotypes (Table 1). Figure 1(b) shows the examples of scanned pictures of 6 strains representing different serotypes. Examples of the same serotype were tested repeatedly and shown to have an identical signal pattern, for example, 5 times for serotype 3 (data not shown). Of 23 strains representing 23-valent vaccine serotype, 18 strains hybridized to all the specific set of probes, and four strains hybridized to almost all the specific set of probes (Table 4). The strain representing serotype 22F may actually belong to serotype group 22F/22A, since this sample failed to hybridize specifically to wchF and wcwA probes but hybridized to the rest of group 22F/22A specific probes. Of the 13 strains representing the 23 vaccine-related serotypes, only 1 isolate (serotype 46), failed to hybridize to a specific probe while the other 12 strains hybridized perfectly. Of the 20 nonvaccine serotypes, 19 strains either hybridized partially to GT-specific probes or did not hybridize to any probes. One strain, representing serotype 23A, hybridized to most of the 23F-specific probe; thus, 23A may be indistinguishable from 23F using GT gene sequences.

Table 4

Microarray results of each strain.

	Serotype	Strain ID	Positive probea	Microarray result
					Assined group
23 serotypes included in 23-valent vaccine	1	ATCC6301	1, 2, 3, 4, 5, 6	Perfectly matched	1
	2	ATCC6302	7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18	Perfectly matched	2
	3	D36	19, 20, 21	Perfectly matched	3
	4	JHK27	22, 23, 24, 25, 26, 27, 28, 29, 30	Perfectly matched	4
	5	ATCC6305	23, 24, 31, 32, 33, 34, 35, 36, 37	Perfectly matched	5
	6B	MSC1047	38, 39, 41, 42, 43	1 probe of group 6A/6B did not hybridized	6A/6B
	7F	ATCC10351	44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58	Perfectly matched	7F/7A
	8	ATCC6308	59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70	Perfectly matched	8
	9V	KD10-11	71, 72, 73, 74, 75, 76, 77, 78, 84, 85, 81, 82, 83	Perfectly matched	9A/9V
	9N	KD01-26	71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83	Perfectly matched	9L/9N
	10A	ATCC8334	86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100	Perfectly matched	10A
	11A	SSI11A/2	101, 102, 103, 104, 105, 106, 107, 108, 109	Perfectly matched	11A/11D
	12F	ATCC6312	11, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124	1 extra probe of group 2 hybridized	12F/12A/12B/44/46
	14	D59	101, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137	Perfectly matched	14
	15B	ATCC10354	125, 128, 131, 135, 138, 139, 140, 141, 142, 143	Perfectly matched	15B/15C
	17F	ATCC6317	144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156	1 extra probe of group 18B/18C hybridized	17F
	18C	ATCC10356	156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166	Perfectly matched	18B/18C
	19F	D33	72, 167, 168,169, 171, 172, 173	Perfectly matched	19F
	19A	D4	71, 73, 74, 169, 170, 171, 172, 173,	1 extra probe of group 19F hybridized	19A
	20	ATCC6320	174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191	Perfectly matched	20
	22F	KD01-23	7, 8, 44, 195, 196, 197, 198, 199, 200	5 probes of group 22F/22A did not hybridized and 1 extra probe of group 7F/7A hybridized	22F/22A
	23F	KD11-15	144, 145, 156, 193, 201, 202, 203, 204, 205, 206, 207	Perfectly matched	23F
	33F	ATCC10370	208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222	Perfectly matched	33F/33A/37
Other serotypes included in 23 groups	6A	MSC1943	38, 39, 40, 41, 42, 43	Perfectly matched	6A/6B
	7A	ATCC6307	44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58	Perfectly matched	7F/7A
	9A	ATCC8333	71, 72, 73, 74, 75, 76, 77, 78, 84, 85, 81, 82, 83	Perfectly matched	9A/9V
	9L	ATCC10349	71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83	Perfectly matched	9L/9N
	11D	SSI11D/1	101, 102, 103, 104, 105, 106, 107, 108, 109	Perfectly matched	11A/11D
	12A	SSI12A/5	110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 274	Perfectly matched	12F/12A/12B/44/46
	12B	SSI12B/1	110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 274	Perfectly matched	12F/12A/12B/44/46
	15C	SSI15C/2	125, 128, 131, 135, 138, 139, 140, 141, 142, 143	Perfectly matched	15B/15C
	18B	ATCC10355	156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166	Perfectly matched	18B/18C
	22A	ATCC10363	7, 48, 49, 192, 193, 194, 195, 196, 197, 198, 199, 200	Perfectly matched	22F/22A
	33A	ATCC8340	208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222	Perfectly matched	33F/33A/37
	44	SSI44/3	110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 274	Perfectly matched	12F/12A/12B/44/46
	46	SSI46/2	110, 111, 112, 113, 114, 115, 116, 117, 119, 120, 121, 122, 123, 124, 274	1 probe of group 12F/12A/12B/44/46 did not hybridized	12F/12A/12B/44/46
Serotypes not included in 23 groups	7B	ATCC10348	143, 155	Partial hybridization	Not included in 23 group
	7C	ATCC10350	none	None hybridization	Not included in 23 group
	10F	ATCC6310	86, 87, 88	Partial hybridization	Not included in 23 group
	10B	SSI10B/2	71, 72, 73, 74, 78, 79, 80, 81, 82, 83	Partial hybridization	Not included in 23 group
	10C	SSI10C/2	71, 72, 73, 74, 75, 76, 77	Partial hybridization	Not included in 23 group
	11F	ATCC6311	103, 104, 105, 106, 107, 108, 109	Partial hybridization	Not included in 23 group
	11B	SSI11B/2	101	Partial hybridization	Not included in 23 group
	11C	ATCC10353	101, 274	Partial hybridization	Not included in 23 group
	15F	ATCC6315	101, 129, 131, 135, 141, 142, 143	Partial hybridization	Not included in 23 group
	15A	ATCC6330	101, 129, 131, 135, 141, 142, 143	Partial hybridization	Not included in 23 group
	17A	SSI17A/2	none	None hybridization	Not included in 23 group
	18F	ATCC6318	144, 145, 156, 157, 158, 159, 162, 163, 164, 165, 166, 193	Partial hybridization	Not included in 23 group
	18A	ATCC10344	144, 145, 156, 158, 160, 161, 162, 164, 165, 166	Partial hybridization	Not included in 23 group
	19B	ATCC10358	72, 167, 168,169, 171, 172	Partial hybridization	Not included in 23 group
	19C	ATCC10359	72,169,171,172	Partial hybridization	Not included in 23 group
	23A	KD12-06	144, 146, 156, 201, 202, 203, 204, 205, 206, 207	1 probe of group 23F did not hybridized	23F
	23B	ATCC10364	7, 46, 202,	Partial hybridization	Not included in 23 group
	33B	ATCC10342	none	None hybridization	Not included in 23 group
	33C	ATCC8339	none	None hybridization	Not included in 23 group
	33D	SSI33D/2	49, 57	Partial hybridization	Not included in 23 group

Explanatory notes: aThe numbers correspond to the spot identifiers given in Tables 2, 3, and Figure 1(a).

4. Discussion

In order to develop a more effective S. pneumoniae vaccine, simple detection methods are required to serotype large numbers of clinical isolates. Conventional serotyping methods using large panels of antisera are labourious and require technical expertise. Our microarray method can determine serotype of a strain at one time and needs no expertise. In addition, the microarray method described here has the potential to be automated. To our knowledge, our report describes the first microarray to utilize GT genes to predict serotype of any bacteria.

Several molecular typing methods have been developed based on serotype-specific sequences [12-21]. Wang et al. [21] described microarray method using wzy and capA genes. Our approach is different in that GT genes were selected as serotype-specific genes. Since GTs catalyze the transfer of the sugar moiety to an acceptor and generate a serotype-specific capsular polysaccharide, detecting GT genes can directly reflect polysaccharide structure. We discovered considerable variability within S. pneumoniae GT genes, which provides groundwork for future investigations into new S. pneumoniae capsular types. Our method using GT genes can not only discriminate serotypes but can give information of the capsular polysaccharide structure.

The DNA microarray described here accurately detects the majority of S. pneumoniae serotypes and serogroups included in the 23-valent vaccine and in the 7, 9, 11, 13-valent conjugate vaccines, which will permit serotype surveillance before and after vaccination. Since 1983, the 23-valent pneumococcal vaccine has been administered to persons in the United States aged >2 years with certain underlying medical conditions or aged >65 years. In 2000, the more effective PCV7, 7-valent pneumococcal conjugate vaccine, which protects against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F was approved for administration [22]. As a result of PCV7, antibiotic-resistant invasive pneumococcal infections have decreased dramatically in young children and older persons [23]; however, an increase in disease associated with serotypes not included in the PCV7 vaccine, has been observed [24, 25]. To address serotype vaccine coverage, the Advisory Committee on Immunization Practices (ACIP) issued recommendations in February 2010 for a newly licensed 13-valent pneumococcal conjugate vaccine (PCV13), which contains the seven serotypes in PCV7 (4, 6B, 9V, 14, 18C, 19F, and 23F) and six additional serotypes (1, 3, 5, 6A, 7F, and 19A) [26]. Taken together, our DNA microarray will be able to monitor serotype prevalence of all vaccine-related serotypes. However, in examining serotype replacement in vaccinated population a further study to distinguish more than 90 serotypes is required and is currently under investigation. Moreover, further study of the reproducibility of the microarray is needed.

5. Conclusion

We developed a S. pneumoniae DNA microarray that identifies GT gene polymorphisms to distinguish capsular types. We believe that our microarray system is more reliable and cost-effective and will help to survey the emergence of new S. pneumoniae serotype.