FEMS Microbiology Letters 233 (2004) 147–152

www.fems-microbiology.org

Virulence of Streptococcus pneumoniae may bedetermined independently of capsular polysaccharide

Y. Mizrachi Nebenzahl a,b,*, N. Porat a,b, S. Lifshitz a,b, S. Novick a,b, A. Levi c,E. Ling a,b, O. Liron a,b, S. Mordechai d, R.K. Sahu d, R. Dagan b

a The Department of Microbiology & Immunology and The Center for Cancer Research, Ben Gurion University of the Negev, Beer-Sheva 84105, Israelb Pediatric Infectious Disease Unit, Soroka University Medical Center, Ben Gurion University of the Negev, Beer-Sheva 84105, Israel

c Department of Epidemiology, Ben Gurion University of the Negev, Beer-Sheva 84105, Israeld Department of Physics, Ben Gurion University of the Negev, Beer-Sheva 84105, Israel

Received 19 August 2003; received in revised form 2 February 2004; accepted 3 February 2004

First published online 20 February 2004

Abstract

Mice were inoculated intranasally with Streptococcus pneumoniae isolates of serotype 14 with different genetic backgrounds (14R,

14DW) and a capsular switch of 14R, strain 9VR (serotype 9V). Inoculation of the mice with 14R and 9VR resulted in 60%

mortality. All the mice survived 14DW inoculation. No differences in lungs’ bacterial loads were found 3 h following inoculation.

Bacterial clearance of 5 logs was observed 48 h after inoculation with 14DW versus within 1 log 48 h after inoculation with 14R and

9VR. No significant differences in bacterial size or the capsular amount could be found between 14R and 14DW. We conclude that

factor(s) in addition to the capsule, contribute to disease outcome.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Streptococcus pneumoniae; Capsular polysaccharide; Virulence factors; Microbial pathogenesis

1. Introduction

Colonization of the nasopharynx by Streptococcus

pneumoniae, although asymptomatic, is a prerequisitefor the development of a clinical infection [1]. The un-

derlying mechanisms that turn the benign state of col-

onization into clinical diseases are poorly understood

[2]. Capsular serotype-specific polysaccharides (PS) were

demonstrated to be a major virulence factor in disease

development following infection [3,4]. Bacterial viru-

lence has been directly correlated to the amount of PS

[5]. Capsular PS’s are able to activate complementleading to C3b deposition and inhibit complement-

mediated opsonophagocytosis [6]. Unencapsulated

mutants exhibit greatly reduced virulence [7]. The anti-

* Corresponding author. Tel.: +11-972-8640-0838; fax: +11-972-

8640-3632.

E-mail address: ymizr@bgumail.bgu.ac.il (Y. Mizrachi Nebenzahl).

0378-1097/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsle.2004.02.003

phagocytic activity of the capsular PS is considered as

pneumococcal major virulence determinant [8].

Specific IgG antibodies directed against the PS cap-

sule are protective against S. pneumoniae infection [9].The current pneumococcal vaccines consist of a mixture

of pure native PS or PS conjugated to a polypeptide

carrier consisting of no more than 23 serotypes [10].

These vaccines are limited in their coverage, and re-

placement diseases with S. pneumoniae serotypes not

included in the vaccines were reported [11].

Understanding the relative contribution of PS and

other bacterial molecules to disease development ishighly important to our understanding of S. pneumoniae

virulence and selection and manipulation of the appro-

priate virulence factor(s) for future vaccine develop-

ment. PS belong to the T cell independent antigens and

as such are ineffective in children under 2 years of age.

However, considerable evidence indicate that T cells

acting in a noncognate manner play a significant role in

the immune response and immunoglobulin production

. Published by Elsevier B.V. All rights reserved.

148 Y. Mizrachi Nebenzahl et al. / FEMS Microbiology Letters 233 (2004) 147–152

in response to PS [12]. Furthermore, dentritic-cells were

shown to mediate in the immune response to PS in an as

yet unknown mechanism [13]. On the other hand pro-

teins are T cell dependent antigens [14], and as such are

efficacious as vaccines in young children. To predictwhich of those components may constitute better puta-

tive future vaccine we have to determine the importance

of the PS and proteins and other bacterial molecules in

virulence and disease development.

In the current study we have tested, in the intranasal

mouse model system, the relative contribution of cap-

sular PS and S. pneumoniae genetic background to dis-

ease outcome.

2. Material and methods

2.1. Bacteria and bacterial growth

The following S. pneumoniae clinical isolates were

used in this study: (1) strain 14DW (serotype 14 ob-tained from Prof. D. Watson Dallas, USA; [15]); (2)

strain 14R isolated from an Israeli child attending day

care center (serotype 14), (3) strain 9VR isolated from

an Israeli child attending day care center and represents

a spontaneous capsular switch of strain 14R. In addition

we have used strain R6 (serotype 2, American Type

Culture Collection; ATCC, Rockville, MD) as control

for the genetic analysis. As control for capsular sizedetermination, we have used strain WU2 (serotype 3)

[7], and the unencapsulated variant of strain 14DW,

strain 14.8 [15].

S. pneumoniae were cultured in Todd–Hewitt broth

(DIFCO laboratories, Detroit, MI, USA) supplemented

with 0.5% yeast extract and grown to mid-late log phase.

The number of CFU were determined in each experi-

ment by plating onto tryptic soy agar supplemented with5% sheep erythrocytes and incubation for 17–18 h at

37 �C under anaerobic conditions.

2.2. Serogrouping and serotyping

Serotyping and serogrouping of S. pneumoniae was

done by means of quellung reaction using antisera

provided by Statens Serum Institute of Copenhagen,Denmark [16].

2.3. Pulsed field gel electrophoresis

Chromosomal DNA fragments, generated by SmaI

and ApaI digestion, were prepared and analyzed as

described elsewhere [17]. A CHEF-DRIII apparatus

(Bio-Rad Laboratories, Richmond, CA) was used forrunning the gels. Running conditions were 23 h at 11.3

�C at 200 V ramped with initial forward time of 5 s and

final forward time of 35 s. Gels were stained with ethi-

dium bromide and photographed. Interpretation of

strains relatedness on the basis of pulsed field gel elec-

trophoresis (PFGE) pattern was performed according to

current consensus criteria [18].

2.4. Mice and intranasal mouse model system

Seven-week-old BALB/c and C57BL/6 mice (ob-

tained from Harlan Laboratories, Israel) were inocu-

lated intranasally with 108 CFU of S. pneumoniae strain

14R (n ¼ 58), the 14R capsular switch serotype 9V se-

rotype (n ¼ 55) or 14DW (n ¼ 72). Survival was moni-

tored daily. Bacterial load was determined by plating theexcised and homogenized lungs onto blood agar plates

at 3 and 48 h post inoculation.

2.5. Quantitation of capsular polysaccharides by ELISA

Quantitation of capsular PS of S. pneumoniae strains

14DW and 14R was determined by sandwich ELISA as

previously described [19]. Four colonies from each wereseparately grown in 10 ml of Todd–Hewitt broth sup-

plemented with 0.5% yeast extract to midlog phase. The

bacterial pellet was sonicated and resuspended in the

original volume. The amount of capsular polysaccharide

was determined in the supernatant and in the bacterial

pellet. Capsular polysaccharide 14 was purchased from

the American Type Culture Collection (Rockland, MD).

Polyclonal rabbit anti serotype 14 and mouse mono-clonal anti serotype 14 were purchased from Staten Se-

rum Institute, Copenhagen, Denmark. The anti mouse

IgG was purchased from Jackson ImmunoResearch

laboratories Inc., West Grove, PA.

2.6. Bacterial size determination by light scattering

spectroscopy

S. pneumoniae strains 14DW and 14R were compared

by light scattering spectroscopy for estimation of bac-

terial size. An He/Ne laser (k ¼ 632:8 nm, 3 mW) was

employed with an ALV-NIBS/HPPS apparatus coupled

with an ALV 5000/ EPP correlator (ALV-GmbH,

Langen, Germany). Fitting was performed by a regu-

larization method which is based on inverse Laplace

transform program CONTIN [20].

2.7. Capsule size determination by Fourier transform

infrared microspectroscopy (FTIR)

Forty ml of the bacterial cultures at OD620 ¼ 0.2 were

fixed with formaldehyde (final concentration 0.25%),

washed three times with saline containing 0.25% form-

aldehyde and the pellet was resuspended in 50 ll saline.One ll of the suspension was spotted on zinc–selenium

slides and air dried for 6 h. Microscopic FTIR mea-

surements in transmission mode were performed using

Y. Mizrachi Nebenzahl et al. / FEMS Microbiology Letters 233 (2004) 147–152 149

the FTIR microscope IRscope II with a mercury–cad-

mium–telluride (MCT) detector, coupled to the FTIR

spectrometer (Bruker Equinox model 55/S, OPUS soft-

ware). Absorbance was measured from a circular area of

100 lm diameter and from regions where the thicknesswas about 10 lm (as seen from the ADC rates) and 128

scans were co-added for each spectrum. The bacterial

spectra were obtained after baseline correction followed

by vector normalization. For each sample at least five

spots were measured and the average spectra calculated.

The spectral intensities at wavenumbers corresponding

to glucose, phosphate and carbohydrates were obtained

after normalization of the region 1600–900 cm�1 to theamide II band at 1542 cm�1 [21,22]. The results reported

are an average of three experiments.

2.8. Statistical methods

Kaplan–Meier survival analysis was used. Statistical

significance was done by 2-tailed Fisher T -test, and the

one way ANOVA (Scheffe) for repeated measurements.

Fig. 1. Pulse field gel electrophoresis (PFGE) analysis of S. pneumoniae

serotype 14DW, 14R 9VR. Chromosomal DNA fragments, generated

by SmaI and ApaI digestion, were analyzed by PFGE. Gels were

stained with Ethidium Bromide and photographed. R6 isolate SmaI

and ApaI digested fragments were used as controls.

Hours100806040200

Prop

ortio

n al

ive

1.1

1.0

.9

.8

.7

.6

.5

.4

Fig. 2. Kaplan–Meier survival analysis of S. pneumoniae (14R 9VR

and 14DW) infection of mice (n ¼ 185). A comparative survival

analysis was performed between seven-week old mice inoculated in-

tranasally with S. pneumoniae strain 14R (–––; n ¼ 58; 28 death events)

to that of mice inoculated with S. pneumoniae serotype 9VR (– – – –;

n ¼ 55; 28 death events) to that of mice inoculated with S. pneumoniae

serotype 14DW ( – - – -; n ¼ 72, no death events). Note: survival of

mice inoculated with strain 14DW different significantly (p < 0:001)

from the survival of mice inoculated with either strain 14R or strain

9VR.

3. Results

3.1. S. pneumoniae genetic background

Pulsed field gel electrophoresis generated by both

SmaI and ApaI confirmed the identity of the geneticbackground of the strain 14R and its spontaneous

capsular switch strain 9VR. Both were found to be

considerably different from the strain 14DW (Fig. 1).

3.2. Intranasal inoculation of the mouse model system

Seven-week-old mice were inoculated intranasally

with 108 CFU of S. pneumoniae strain 9VR or 14R or14DW. Intranasal inoculation with strain 14R or its

capsular switch 9VR resulted in mortality rates of 50%

within 2 days and 60% within 4 days with no significant

differences between the two. In contrast, all mice sur-

vived intranasal inoculation with strain 14DW. Inocu-

lation of the mice with S. pneumoniae strain 14DW

differed significantly (Kaplan–Meier analysis, p < 0:001)from the infection outcome with S. pneumoniae strain14R and its capsular switch serotype 9VR (Fig. 2). To

exclude the possibility that disease outcome results from

the genetic background of the mice we have used C57BL/

6 and BALB/c mice. No significant differences could be

found between the BALB/c and the C57BL/6 mice.

Animals were sacrificed at 3 h (9VR n ¼ 11; 14R

n ¼ 20; 14DW n ¼ 20) and 48 h (9V n ¼ 8; 14R n ¼ 14;

14DW n ¼ 30) following intranasal inoculation. Lungswere excised and a homogenate of the right lung was

plated on blood agar plates. No significant differences

could be found between the BALB/c and the C57BL/6

150 Y. Mizrachi Nebenzahl et al. / FEMS Microbiology Letters 233 (2004) 147–152

inbred mice in the extent of lung colonization and thus

the results were pooled.

The extent of the colonization of the lungs 3 h fol-

lowing intranasal inoculation of the mice with either S.

pneumoniae strain 14R, or the capsular switch serotype9VR, or 14DW did not differ significantly (one way

ANOVA p > 0:05; Fig. 3). However, 48 h following

inoculation, a spontaneous bacterial clearance could be

observed in S. pneumoniae strain 14DW with a 5 logs

reduction in bacterial load in comparison to the extent

of the bacterial load in the lungs found at 3 h post in-

oculation (p < 0:001; Fig. 3). In contrast, only within

one log reduction in the bacterial load in the lungs,could be observed 48 h following inoculation with S.

pneumoniae strain 14R and its capsular switch serotype

9VR in comparison to bacterial load in the lungs found

at 3 h following inoculation (p < 0:027 and p < 0:021,respectively). Bacterial load in the lungs of mice ana-

lyzed 48 h following intranasal inoculation with S.

pneumoniae serotype 14DW differed significantly from

the bacterial load found in the lungs of mice inoculatedintranasally with either S. pneumoniae strain 14R or

9VR (p < 0:001 in both; Fig. 3). No significant differ-

ences could be found between the bacterial load in the

lungs of mice inoculated with S. pneumoniae strain 14R

and 9V 48 h post inoculation (p ¼ 1:00).

3.3. Determination of capsular sized

Quantification of the amount of capsular PS was

performed using sandwich ELISA. The amount of

9 V (3h) 9V (48h) 14R (3h) 14R(48h) 14DW(3h) 14DW (48h)

Bacterial strain (time post-inoculation)

0

102

103

104

105

106

107

108

Log

CF

U in

the

lung

+/-

SE

M

Fig. 3. Bacterial load in the lungs. Seven-week-old mice were inocu-

lated intranasally with 5� 108 CFU of S. pneumoniae strain 14R or

9VR or 14DW. Animals were sacrificed at 3 h (9V n ¼ 11; 14R n ¼ 20;

14DW n ¼ 20) and 48 h (9V n ¼ 8; 14R n ¼ 14; 14DW n ¼ 30) fol-

lowing inoculation. Lungs were excised and a homogenate of the right

lung was plated on blood agar plates. No significant differences in

bacterial load in the lungs could be found 3 h following inoculation

(One way ANOVA, p > 0:05). Forty eight hours post inoculation

bacterial load in the lungs of S. pneumoniae strain 14DW differed

significantly from the bacterial load in strain 9VR (< 0:001) and from

the bacterial load strain 14R (p < 0:001).

capsular PS was determined in four colonies from each,

S. pneumoniae strain 14DW and strain 14R, in the su-

pernatant and the washed bacterial pellet and the results

were combined and normalized to the amount of protein

in the pellet. In S. pneumoniae 14R we have found1051.15� 328.64 ng PS/lg protein and in S. pneumoniae

14DW 530� 329 ng PS/lg protein. No significant dif-

ferences between the two type 14 strains could be found

(Fisher T -test, p ¼ 0:66).

3.4. Bacterial size determination by light scattering

spectroscopy

We have measured bacterial size of 14DW and 14R,

using light scattering spectroscopy. The bacterial iso-

lates were grown on blood agar plates and five colonies

of each were analyzed. Strains, 14DW and 14R exhib-

ited a mean radius of 389� 20 nm each (Fisher T -test;p ¼ 0:987).

3.5. Capsule size determination by Fourier transform

infrared microspectroscopy

An additional method was employed to confirm

strain size. Fourier transform infrared microspectros-

copy (FTIR-MSP) is a non-destructive technique where

whole cells can be used for spectral characterization

based on their biochemical composition. The carbohy-

drate content determined from the FTIR spectral datashowed significant differences between strain WU2 and

all the other strains used in this study (ANOVA test

1600 1400 1200 1000 8000.0

0.5

1.0

1.5

2.0

14R

14DW

9V

14.8

WU2

Abs

orba

nce

( A

.U.)

Wavenumber ( cm )-1

Fig. 4. Capsule size determination by Fourier transform infrared

microspectroscopy (FTIR-MSP). FTIR-MSP of the various bacterial

strains in the region 800–1600 cm�1 after normalization to the amide II

band at 1542 cm�1. The spectra are the averages of 128 co-added

scans. It should be noted that significant differences exist between

strain WU2 and all the other strains used in this study (ANOVA test

p < 0:001). Similarly, the unencapsulated variant used, strains 14.8

differed significantly from strains 14DW, 14R and 9VR (p < 0:001).

However, there was no statistical significant difference between

S. pneumoniae strains 14DW and 14R (p > 0:005).

Y. Mizrachi Nebenzahl et al. / FEMS Microbiology Letters 233 (2004) 147–152 151

p < 0:001). Similarly, the unencapsulated variant used,

strains 14.8 differed significantly from strains 14DW,

14R and 9VR (p < 0:001). However, there was no sta-

tistical significant difference between S. pneumoniae

strains 14DW, 14R and 9VR (Fig. 4).

4. Discussion

We had the opportunity to study infection induced in

the intranasal mouse model system by a clinical S.

pneumoniae isolate and its capsular switch carrying an

identical genetic background. We compared the abilityof these two isolates (strains 14R and its capsular switch

9VR) to cause pneumonia and sepsis following intra-

nasal infection in a mouse model system to that of a

serotype 14 clinical isolate (strain 14DW) with a differ-

ent genetic background.

The relative contribution of capsular PS and the non-

capsular components of S. pneumoniae to virulence are

not fully understood. Using the intraperitoneal route ofmice infection, transformation of type 2 recipient strain

with a type 3 capsule did not alter the extent of viru-

lence, which resembled the type 2 strain. However,

transformation of a serotype 5 and 6B with a type 3

capsule attenuated the virulence of the transformed

bacteria [23]. In another study, using the intraperitoneal

route of infection of mice, variation in capsule pheno-

type determined the survival rates of mice rather thanthe similarities in the genetic background [24]. These

studies may shed light on the antiphagocytic activity,

among others, of S. pneumoniae capsule. However, they

fail to explain relative importance of the capsule or the

bacterial genetic background to disease development

during the nasopharyngeal infection, which is more

common in the case of S. pneumoniae.

Recently, Kadioglu et al. [25] have shown, using achimeric mutant in which capsule type 3 was inserted

into type 2 bacteria, that the combination of capsule and

genetic background were both important, and the in-

fluence of the combination varied with site of infection.

The wild type serotype 3 (A66) and the capsule switch

colonized the outbred mice, used in this study, to the

same extent following intranasal inoculation, but did

not colonize the lungs as efficiently. Furthermore, thechimeric bacterium was avirulent in comparison to its

parental strains. It should be noted that in this study, the

relative amount of capsule in the parental and the chi-

meric strains was not determined.

In a recent study in young children [26], S. pneumo-

niae isolates from invasive diseases were compared with

those nasopharyngeally carried by healthy children. This

was done using multilocus typing and capsular sero-typing. It was found that the capsule serotype seem to be

the predominant factor in determining invasive disease.

It should be noted that this study was performed in one

community. Furthermore, this does not preclude the

contribution of other virulence factors to diseases de-

velopment. However, the extent of contribution to vir-

ulence of the different factors has to await further

understanding of S. pneumoniae pathogenic process.In the current study similarities in genetic back-

ground in S. pneumoniae strain 14R and its capsular

switch 9VR coincided with similarities in disease out-

come following intranasal inoculation, independent of

the capsular type. A genetically different serotype 14

strain (14DW), with a similar bacterial size carrying a

similar amount of capsule, did not inflict a lethal disease

following intranasal inoculation. Moreover, the clear-ance of the 14DW strain from the lungs of the mice was

more pronounce and coincided with the ability of the

mice to recover spontaneously from the infection.

To circumvent the possibility that the immunogenetic

background of the mice may have affected the differ-

ences in the disease outcome, we have used both the

C57BL/6 and the BALB/c mice in our studies. No sig-

nificant differences in disease outcome could be notedalbeit the well documented differences in the immune

response of these two inbred mouse strains [27]. This

also suggests that the results obtained were bacterial

rather than host dependent.

The results presented in the current study further

suggest that the extent of bacterial virulence cannot be

predicted from its capsular type only. In some circum-

stances factor(s) other than capsular PS may be impor-tant in determining pneumococcal disease outcome.

Among those factor may be proteins [28], lipids and

differences in the cell wall components. We are currently

exploring the involvement of surface proteins and

membrane lipids on S. pneumoniae virulence. General-

ization and extrapolation of the results obtained in our

mouse model to the human disease outcome will have to

await further studies.

Acknowledgements

This study was partially supported by a grant from

the Israeli MOH #4776 to YMB and by a grant from the

Centre of Emerging Diseases #2506 to YMB. Special

thanks to Ms. Ronit Trefler and Marilon Shagan and

Mr. Azmi Adawi for their excellent technical help.

Thanks to Prof. M. Gotlib and Dr. S. Vanunu for their

help in the light spectroscopy analysis.

References

[1] Jenkinson, H.F. and Lamont, R.J. (1997) Streptococcal adhesion

and colonization. Crit. Rev. Oral Biol. Med. 8, 175–200.

[2] Tuomanen, E.I. (1997) The biology of pneumococcal infection.

Pediatr. Res. 42, 253–258.

152 Y. Mizrachi Nebenzahl et al. / FEMS Microbiology Letters 233 (2004) 147–152

[3] Magee, A.D. and Yother, J. (2001) Requirement for capsule in

colonization by Streptococcus pneumoniae. Infect. Immunol. 69,

3755–3761.

[4] Avery, O.T. and Goebel, W.F. (1929) Chemo-immunologic

studies on conjugate carbohydrate-proteins. II Immunological

specificity synthetic sugar-protein antigens. J. Exp. Med. 50, 533–

550.

[5] Kim, J.O. and Weiser, J.N. (1998) Association of intrastrain phase

variation in quantity of capsular polysaccharide and teichoic acid

with virulence of Streptococcus pneumoniae. J. Infect. Des. 177,

368–377.

[6] Brown, E.J. (1985) Interaction of gram-positive organisms with

complement. Curr. Top Microbiol. Immunol. 121, 159–187.

[7] Watson, D.A. and Musher, D.M. (1990) Interruption of capsule

production in Streptococcus pneumoniae serotype 3 by insertion of

transposon Tn916. Infect. Immunol. 58, 3135–3138.

[8] Tuomanen, E.I. and Masure, R.H. (2000) Molecular and cellular

biology of pneumococcal infection. In: Streptococcus pneumoniae

molecular biology and mechanism of disease, pp. 295–308. Mary

Ann Liebert, New York.

[9] Lindberg, A.A. (1999) Polysides (encapsulated bacteria). C.R.

Acad. Sci. III 322, 925–932.

[10] Dagan, R., Muallem, R., Melamed, O., Leroy, O. and Yagupsky,

P. (1997) Reduction of pneumococcal nasopharyngeal carriage in

early infancy after immunization with tetravalent pneumococcal

vaccine conjugated to either tetanus toxoid or diphtheria toxoid.

Pediatr. Infect Dis. J. 16, 1060–1064.

[11] Hausdorff, W.P., Bryant, J., Kloek, C., Paradiso, P.R. and Siber,

G.R. (2000) The contribution of specific pneumococcal serogroups

to different disease manifestations: Implication for conjugate

vaccine formulation and use. Part II. Clin. Infect. Dis. 30, 122–

140.

[12] Mond, J.J., Lees, A. and Snapper, C.M. (1995) T-cell-independent

antigen type2. Annu. Rev. Immunol. 13, 655–692.

[13] Colino, J., Shen, Y. and Snapper, C.M. (2002) Dentritic cells

pulsed with intact Streptococcus pneumoniae elicit both protein-

and polysaccharide specific immunoglobulins isotype response in

vivo through distinct mechanisms. J. Exp. Med. 195, 1–13.

[14] Svensson, M., Stockinger, B. and Wick, M.J. (1997) Bone

marrow-derived dentritic cells can process bacteria for MHC-II

and MHC-II presentation to T cells. J. Immunol. 158, 4229–4236.

[15] Watson, D.A., Kapur, V., Musher, D.M., Jacobson, J.W. and

Musser, J.M. (1995) Identification, cloning and sequencing of

DNA essential for encapsulation of Streptococcus pneumoniae.

Curr. Microbiol. 31, 251–259.

[16] Austrian, R. (1976) The quellung reaction, a neglected microbi-

ological technique. Mt Sinai J. Med. 43, 699–709.

[17] Soares, S., Kristinsson, K.G., Musser, J.M. and Tomasz, A.

(1993) Evidence for the introduction of a multiresistant clone of

serotype 6B Streptococcus pneumoniae from Spain to Iceland in

the late 1980s. J. Infect. Dis. 168, 158–163.

[18] Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A.,

Murray, B.E., Persing, D.H. and Swaminathan, B. (1995) Inter-

preting chromosomal DNA restriction patterns produced by

pulsed-field gel electrophoresis: criteria for bacterial strain typing.

J. Clin. Microbiol. 33, 2233–2239.

[19] Roche, J.R. and Moxon, E.R. (1995) Phenotype variation in

Haemophilus influenza: the interrelationship of colony opacity,

capsule and lipopolysaccharide. Microb. Pathog. 18, 129–140.

[20] Provencher, Stephen W. (1982) CONTIN: a general purpose

constrained regularization program. Comput. Phys. Commun. 27,

229–242.

[21] Naumann, D. (2001) FT-Infrared and FT-Raman spectroscopy in

biochemical research. In: Infrared and Raman spectroscopy of

biological materials (Gremlich, H.-U. and Yan, B., Eds.) (Ed.),.

In: Practical Spectroscopy Series, vol. 24, pp. 323–377. Marcel

Dekker, Inc, New York (Chapter 9).

[22] Parker, F.S. (1971) Application of Infrared Spectroscopy in

Biochemistry, Biology and Medicine. Plenum, New York.

[23] Kelly, T., Dillard, J.P. and Yother, J. (1994) Effect of switching

capsular type on virulence of Streptococcus pneumoniae. Infect.

Immunol. 62, 1813–1819.

[24] Nesin, M., Ramirez, M. and Tomasz, A. (1998) Capsular

transformation of a multidrug resistant Streptococcus pneumoniae

in vivo. J. Infect Dis. 177, 707–713.

[25] Kadioglu, A., Taylor, S., Iannelli, F., Pozzi, G., Mitchell, T.J. and

Andrew, P.W. (2002) Upper and lower respiratory tract infection

by streptococcus pneumoniae is affected by pneumolysin deficiency

and differences in capsule type. Infect. Immunol. 70, 2886–2890.

[26] Brueggemann, A.B., Griffiths, D.T., Meats, E., Peto, T., Crook,

D.W. and Spratt, B.G. (2003) Clonal relationships between

invasive and carriage Streptococcus pneumoniae and serotype-

and clone-specific differences in invasive disease potential. J. Infect

Dis. 187, 1424–1432.

[27] Ulett, G., Ketheesan, N. and Hirst, R. (2000) Cytokine gene

expression in innately susceptible BALB/c mice and relatively

resistant C57BL/6 mice during infection with virulent Burkholde-

ria pseudomallei. Infect. Immunol. 68, 2034–2042.

[28] Jedrzejas, M.J. (2001) Pneumococcal virulence factors: structure

and function. Microb. Mol. Biol. Rev. 65, 187–207.