HISTORICAL ARTICLE

A Brief History of the Pneumococcus in Biomedical Research: A Panoply of
Scientific Discovery

David A. Wqatson, Daniel M. Musher,
James W. Jacobson, and Jan Verhoef

From the Veterans Afsairs Medical Center, Baylor College of Medicine.
   and the University of Houston, Houston. Texas; and Academic
           Hospital Utrechl. Utrecht, The Netherlands

Because of its prominence as a cause of disease in humans, Streptococcuspneumoniae has been
the subject of intensive investigation at both the clinical level and the basic scientific level during
the past century. In a number of instances, these studies have resulted in important progress
toward the comprehension of basic biological principles. The areas advanced by studies of the
pneumococcus include an understanding of the concept of pathogenesis of infectious disease; the
development of Gram's stain for identification of bacteria in specimens from patients; the eluci-
dation of the role of the bacterial capsule in resistance to phagocytosis by cells of the host's
immune system; the demonstration that molecules other than proteins are capable of eliciting the
host's humoral immune responses and later, by extension, that isolated bacterial exopolysacchar-
ides can be used safely and effectively as vaccines in humans; the documentation of the efficacy of
penicillin; the collection of conclusive evidence that DNA encodes genetic information; and the
investigation of putative proteinaceous virulence factors.

Data acquired during the course of clinical investigations
can often provide the answers to basic biological questions if
subjected to critical analysis by insightful researchers. Ample
illustrations of this point are found in the abundant reports
of investigations involving Streptococcus pneumoniae, an or-
ganism of unquestioned clinical importance. Since a variety
of recent reviews have focused on different aspects of pneu-
mococcal research, we will not attempt an exhaustive sum-
mary here; rather, we will direct the reader to detailed re-
views where appropriate. In this review we will focus on the
ways in which studies of this pathogen have been central to
several of the most profoundly influential biological findings
of the past 110 years.

Description of the Organism and Demonstration of Its
Virulence

In 188 1 two microbiologists, George M. Sternberg in the
United States and Louis Pasteur in France, independently
described roughly lancet-shaped pairs of coccoid bacteria in

Received 9 November 1992; revised 2 April 1993.
Portions of this paper were included in the doctoral dissertation
of D. A. W.

Financial support: This work was funded by the Department of Veterans
Affairs.

Reprints or correspondence: Dr. David A. Watson. Department of Veteri-
nary and Microbiological Sciences, North Dakota State University, Van Es
Hall, P.O. Box 5406, Fargo, North Dakota 58 105.
Clinical Infectious Diseases 1993;17:913-24
0 1993 by The University of Chicago. All rights reserved.
lO58-4838/93/1705-0058$02.00

human saliva. Pasteur [ 1, 21 and Stemberg [3, 41 each in-
jected human saliva into rabbits; Pasteur used saliva from a
child who had died of rabies, while Sternberg used his own
saliva. Both researchers subsequently recovered diplococci
from the blood of these rabbits.

Previous reports identifying slightly elongated diplococci
existed in the literature [5,6], but only Sternberg and Pasteur
demonstrated the pathogenic potential of these bacteria in
animals. In fact, each researcher had described the same or-
ganism; it was named Microbe septicemique du salive by Pas-
teur [2] and Micrococcus pasteuri by Sternberg [7]. By 1886
this organism was being referred to as Pneumococcus by
Fraenkel [S] because of its propensity to cause pulmonary
disease. It was renamed Diplococcus pneumoniae in I920 [9]
-a designation obviously referring to pairs of cocci causing
pneumonia. This epithet was first suggested by Weichsel-
baum in 1886 [ lo- 131 in a series of case reports on the caus-
ative agent ofwhat was then called croupous pneumonia; he
also referred to pneumococci as "kapsel kokken." It was not
until 1974, however, that the pneumococcus was given its
present name, Streptococcus pneumoniae [ 141, primarily on
the basis of its characteristic growth as chains of cocci in
liquid media.

The causative role of this organism in human lobar pneu-
monia was firmly established in the early 1880s by a number
of investigators [ 15- 181; later in that same decade, the pneu-
mococcus was clearly demonstrated to be a cause ofmeningi-
tis [ 191 and otitis media [20]. Robert Austrian, an influential
researcher on pneumococcal vaccines and a noted historian
of the pneumococcus, has written two excellent reviews on
the latter subject [2 1, 221.


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Watson et al.                  CID 1993; I7 (November)

Gram's Stain

Also during the 188Os, Christian Gram [23] was experi-
menting in the laboratory of Friedlgnder [22] with tech-
niques for visualization of bacteria in pathological speci-
mens. Gram examined sections of lung tissue from patients
who had died of pneumonia; he exposed the specimens se-
quentially to aniline-gentian violet; a weak solution of io-
dine; ethanol; and Bismarck brown or vesuvin. Gram found
that these sections contained many pairs of slightly elon-
gated cocci that retained the dark aniline-gentian violet
stain. He referred to these organisms as "the cocci of crou-
pous pneumonia." The failure of other bacteria in Gram's
specimens to retain the aniline-gentian violet demonstrated
a phenomenon that would become one of the cornerstones
of clinical microbiology-namely, that nearly all clinically
important bacteria are either gram-positive or gram-negative.
In fact (as discussed by Austrian [21]), in some of the lung
sections described above, Gram saw an encapsulated bacte-
rium that did not retain the aniline-gentian violet and that
caused pneumonia (Klebsiella pneumoniae, or Friedlgnder's
bacillus). This observation, had he fully appreciated it, could
have forestalled an acrimonious debate between Fraenkel
and Friedlgnder over the etiology of lobar pneumonia; in
fact, each was correct [2 11.

The pneumococcus, therefore, was one of the first patho-
genic bacteria observed during the development of Gram's
stain, a bacteriologic tool that is still in everyday use more
than a century after its original description.

Humoral Immunity, Bacterial Capsules, and
Phagocytosis

After the early descriptions of the role of the pneumococ-
cus in disease, Klemperer and Klemperer [24, 251 showed
that serum from rabbits injected with heat-killed pneumo-
cocci or with filtrates of broth cultures contained factors that
conferred immunity to reinfection with the same strain but
not necessarily to infection with different clinical isolates.
More important, rabbits were protected against primary
pneumococcal infection by infusion of serum from a previ-
ously immunized animal [24, 251. Issaeff [26] demonstrated
shortly thereafter that this protective serum was not directly
bactericidal but that it did promote uptake of pneumococci
by phagocytic cells of the immune system. Earlier, the well-
known immunologist Eli Metchnikoff had observed pneu-
mococcal agglutination in antisera [27], but he apparently
did not make the connection between this agglutinating fac-
tor and the promotion of phagocytosis. This point is ironic,
in that Metchnikoffwas the first to describe the phenomenon
of phagocytosis. In any event, S. pneumoniae was the organ-
ism used to document the protection of animals by active
immunization and the presence of the protective factor in
serum.

Figure 1.  Upper panel: Reactivity of S. pneumoniae serotype 8
(ATCC 6308) with antiserum pools A, B, and C. Specific agglutina-
tion is evident for pool B, which contains antibody specific for
serotypes 3,4, and 8, and group 19. Lowerpanel: Specific reactivity
of the same strain with antiserum to serotype 8 polysaccharide.
Pneumococci were stained with ethidium bromide, washed twice
with PBS, resuspended at a concentration of - 5 X IO'cfu/mL, and
mixed I : 1 with the indicated antisera.

At the turn of the century, Neufeld demonstrated both
macroscopic agglutination and microscopically visible, spe-
cific swelling (queflung in his native German) of the external
capsule upon the addition of specific antiserum to a suspen-
sion of pneumococci [28]. For most pneumococcal sero-
types, homologous rabbit polyclonal antiserum mixed in
equal parts with a cloudy suspension of bacteria (N 10' cfu)
results in macroscopically visible bacterial clumping (figure
I), thereby providing a simple method of serotyping. Bile
solubility testing, the quellung and agglutination reactions,
and additional techniques are lucidly discussed in a classic
review of the laboratory identification of pneumococci by
Lund [29].

,

The apparent discrepancy between humoral and cellular
immunity was resolved in 1904, when Neufeld and Rimpau
[30] showed that ingestion of pneumococci by white blood
cells was greatly facilitated by preexposure of the bacteria-
but not the white cells-to serum from a previously immu-
nized animal. The phenomenon they demonstrated was
what we now call opsonization (from the Greek word for
"preparing food"), in which the coating of bacteria with
complement components and immunoglobulins leads to Fc
receptor-mediated uptake by phagocytic cells.

Definitive proof of the critical importance ofthe capsule to
virulence was established in a pair of papers printed back to
back in the Journal of Experimental Medicine in 193 1, In the
first paper Rene Dubos and Oswald Avery showed that an
enzyme obtained from a soil bacillus removed the serotype 3
capsular polysaccharide [31]. In the second paper [32] these


CID 1993; I7 (November)

The Pneumococcus in Biomedical Research

915

investigators demonstrated the protection of mice by the en-
zyme against otherwise-fatal challenge with S. pneumoniae
serotype 3. This enzyme was later shown by Francis et al.
[33] to provide the same protection to the Java monkey.

It is clear from the foregoing discussion that, even without
knowledge of the specific structure or mode of action of anti-
bodies, early investigators were well aware of the presence
and importance of this serum component. Less well studied
in the early 1930s was the possible role ofnonimmunoglobu-
lin serum molecules in opsonophagocytosis of pneumococci.
While Ward and Enders [34] first demonstrated the necessity
for such a factor in 1933, little additional work was com-
pleted until 1969, when Johnston et al. [35] outlined the
effect of complement in increasing the rate of pneumococcal
phagocytosis. Winkelstein and (later) Hosea, Brown, and
other researchers specifically delineated the locations and
mechanisms of activation of the classical and alternative
pathways of complement by encapsulated pneumococci,
leading to phagocytosis (see [36] for references). These stud-
ies helped to clarify the relative contributions of immuno-
globulin and complement opsonins to the opsonophagocyto-
sis of encapsulated pathogenic bacteria. The subject has been
reviewed by Winkelstein [36] and-quite recently-by Jan-
off et al. [ 371 as part of a broader discussion of pneumococcal
disease during infection due to human immunodeficiency
virus.

The Concept of Serotyping

The discovery that the injection of pneumococci into rab-
bits had an immunizing effect facilitated the development of
an elementary typing system for this bacterial species. Neu-
feld and Haendel [38] classified isolates from patients with
confirmed pneumococcal pneumonia into two groups on the
basis of whether or not they killed mice previously immu-
nized with pneumococcal isolates referred to as type I or type
II. The authors correlated these results with those obtained
in agglutination reactions. Three years later Dochez and Gil-
lespie [39] extended these groupings to include three distinct
pneumococcal serotypes as well as a fourth group that was
heterogeneous. All isolates ofthe first three serotypes reacted
with antiserum to any other organism of the same serotype.
In contrast, each member of the fourth cluster failed to react
with antisera to the first three serotypes but instead tended to
react only with antiserum produced by immunization of a
rabbit with that specific isolate. Lister [40, 411, working in
South Africa, confirmed the validity of this typing system
and showed that virulent strains unrelated to the American
strains studied by Dochez and Gillespie existed in South
Africa.

It is worth noting that the third pneumococcal serotype to
be established was phenotypically distinct from types 1 and 2
and from the group 4 isolates; when grown on solid agar, it
produced colonies that were noticeably larger, more mucoid,

and more iridescent than those produced by serotype 1 or 2
or by group 4. In fact, for some time, what we now refer to as
S. pneumoniae serotype 3 was considered to be a separate
species known as Pneumococcus mucosus [42]. It is now
known, however, that serotype 37 also exhibits a highly mu-
coid phenotype and thus is macroscopically indistinguish-
able from serotype 3 on blood agar plates [43]; moreover, on
rare occasions, we have observed this phenotype among clin-
ical isolates of serotypes 6A and 19F (authors' unpublished
observations). The detection of recurring reactive types (sero-
types) among group 4 pneumococci eventually led to the
identification of 85 distinct serotypes [44], largely through
the efforts of Cooper, Eddy, March, and Lund before 1960.
Lund [29] beautifully reviewed the history of these studies.
An excellent review of the immunogenicity and immuno-
chemistry of pneumococcal capsular polysaccharides has re-
cently been published by van Dam and associates [44].

Polysaccharides as Capsular Material

While working at the Rockefeller Institute in New York
City in 19 17, Dochez and Avery [45] described a soluble
specific substance they had found in serum and urine from
patients with lobar pneumonia and in blood from animals
experimentally infected with pneumococci; this substance
formed a precipitate with specific antiserum to the homolo-
gous pneumococcus. By identifying this substance-which
comprised the pneumococcal cell envelope-as a complex
carbohydrate or polysaccharide, Heidelberger and Avery
[46] unambiguously established that the capsular polysaccha-
ride was the factor responsible for serological reactivity. Of
the pneumococcal cell, Heidelberger later concluded [47]:
"[Tlhere is disposed at its periphery a highly reactive sub-
stance upon which type specificity depends." Heidelberger
and colleagues further showed that this capsule was anti-
genie; that is, the complex carbohydrate composing this cov-
ering induced immunity in mice that protected these animals
from lethal infection upon subsequent pneumococcal chal-
lenge. Before this seminal observation was reported, it had
been widely believed that only proteins were capable ofelicit-
ing an immune response [48].

,

Vaccine Studies

Even before the demonstration of the immunogenicity of
the bacterial capsular polysaccharide, studies begun in I9 I 1
by Sir Almroth E. Wright and colleagues [49]-with South
African gold miners as test subjects-suggested that inocula-
tion of whole killed pneumococci might elicit protection
against pneumococcal infection in human beings [50]. In
this work Wright followed the principles of study he had
already used with reasonable success in vaccinating subjects
against typhoid fever [5 I]. Unfortunately, the results he ob-
tained with pneumococcal vaccine did not convince the sci-


916

Watson et al.

CID 1993; 17 (November)

entific community of its efficacy. The problem lay in the
failure to include both pneumococcal serotypes known at
that time and in the use of an inadequate vaccine dosage [22]
because of the discomfort associated with the injection of
relatively large inocula of whole killed pneumococci.

In 1926 Felton and Baily [52] described the separation of
capsular polysaccharides and showed that the resulting mate-
rial, called "soluble specific substance," was the subcellular
fraction responsible for conveying immunity. This work
opened the door for Francis and Tillett [53] and Finland and
co-workers [54-571 to conduct a number of studies (during
the 1930s and 1940s) of the effectiveness of vaccines aimed
at the prevention of pneumococcal disease. In 1937 Felton's
capsular material was used successfully in a program of mass
vaccination to abort an outbreak of pneumonia at a state
hospital [58]; this was the first instance in which active vacci-
nation with a relevant subcellular bacterial fraction had been
used for such a purpose. Besides Finland, other pioneers in
the field at this time included Felton himself 1591, MacLeod
and colleagues [60], and Heidelberger and associates [61];
each investigator or group of investigators showed that
healthy adult volunteers were protected against pneumococ-
cal infection by vaccines that stimulated the immune system
to produce antibodies to the pneumococcus. Kaufman [62]
demonstrated that pneumococcal vaccines containing two
and later three type-specific polysaccharides (i.e., bivalent
and trivalent vaccines) were efficacious in an elderly cohort.
These studies led to the licensing of hexavalent polysaccha-
ride vaccines for human use after World War II. However,
these vaccines were not used by physicians at that time be-
cause many believed that newly available drugs constituted a
more effective means of dealing with pneumococcal disease;
as a result, the vaccines were withdrawn from the market [22].

Interest in pneumococcal polysaccharide vaccines was re-
vived in the mid- 1960s largely because of the efforts of Rob-
ert Austrian. Work on a multivalent vaccine containing the
polysaccharide components of each of the 14 most common
pneumococcal serotypes (which caused some 80% of cases of
pneumococcal disease) began in 1967 and culminated in the
introduction of a ICvalent vaccine in 1977. This advance
followed studies by Austrian et al. [63, 641 in which such a
vaccine was efficacious in certain populations with high at-
tack rates of pneumococcal pneumonia. A 23-valent vaccine
containing an even larger percentage of the pneumococcal
serotypes commonly causing disease was introduced in I983
[65] and is the subject of recent reviews [66, 671 and com-
ment [68]. A number of studies have evaluated the efficacy
of this vaccine [69-711, and all have yielded values in the
range of 55%65%. The most recent of these reports also
showed that the age and immune status of the patient as well
as the interval since vaccination all figure significantly into
the equation [72]. The degree of efficacy has not been uni-
form in all populations, however, with particularly low suc-
cess rates among very young children [73], debilitated el-

derly persons [74], or individuals whose immune systems are
compromised [72, 751; production of a vaccine that is effi-
cacious in these high-risk groups remains a cherished but
elusive goal. Pneumococcal polysaccharides of several sero-
types have been conjugated to carrier proteins [76-791, a
technique previously used with great success for Haemophi-
lus injuenzae type b polyribosyl ribitol phosphate. A number
of clinical trials of pneumococcal conjugate vaccines are in
progress; to date, the results have been generally favorable
[72], though not unequivocally so (D. M. Musher, M. C.
Rodriguez-Barradas, J. E. Groover, and D. A. Watson, un-
published observations). While much remains to be accom-
plished in this area, Broome and Breiman [68] point out that
the current 23-valent vaccine can greatly reduce the number
of cases of bacteremic pneumococcal infections and should
therefore be more widely administered to the persons for
whom its use is indicated.

Chemotherapy

In 19 11 Morganroth and Levy [80] showed that a quinine
derivative, ethylhydrocupreine (also known as optochin), in-
hibited the growth of pneumococci but not of clinically re-
lated organisms. The use of optochin by Morganroth and
Kaufmann [8 l] to treat experimentally infected mice is one
of the first examples of the use of a specific antimicrobial
agent as therapy for a serious bacterial infection-and, in
fact, of any highly specific compound as therapy for any in-
fection. Quinine had previously been evaluated for the treat-
ment of pneumococcal pneumonia in humans [50]; the min-
imal success of this effort contrasted with the great
importance of quinine in the treatment of malaria. Mor-
ganroth and Kaufmann showed that pneumococci rapidly
became resistant to clinically achievable doses of optochin,
possibly through the acquisition of a single point mutation,
as our recent data suggest [82]. In addition, optochin had
only a narrow window of effectiveness between therapeutic
and toxic dosages [83]; its use was rapidly abandoned due to
its optic toxicity [ 841.

.

Serotherapy

During the 1930s two important new approaches to ther-
apy were developed, at least in part through their application
to pneumococcal infection. The first approach-the infusion
of pneumococcal antiserum produced in animals for the
treatment of active pneumococcal infection in humans [85]
-had been shown much earlier to be effective in animals
[24,25]. In the last decade ofthe nineteenth century, numer-
ous investigators had obtained mixed results with immune
serum from a variety of animal sources [50]. Interest in this
approach was probably fueled by the successful reduction in
mortality from diphtheria by the same basic technique. How-
ever, the underlying principle was quite different in the latter


CID 1993: I7 (November)

The Pneumococcus in Biomedical Research

917

case: serotherapy for diphtheria involved an antiserum to the
toxin, whereas serotherapy for pneumococcal infection was
aimed at the transfer of antibody that would opsonize the
infecting organism and therefore eradicate it from the host. It
was not until the 1920s-when serotypes began to be recog-
nized, when antisera were standardized according to sero-
type, and when sera from repeatedly sensitized horses were
used-that consistently good results were first reported [86].
Sera from patients who had recovered from pneumonia were
theoretically preferable to those from animals because of the
reduced risk of serum sickness; unfortunately, the potency of
these preparations of human serum was inferior, and their
use was abandoned [87].

Use of Antimocrobii Agents

The second new approach to therapy was the administra-
tion of defined chemotherapeutic agents-first sulfanila-
mide and later penicillin.

Sulfanilamide.  Among the earliest uses of the antimicro-
bial compound sulfanilamide was that for the treatment of
pneumococcal pneumonia, although the frequency with
which this option was selected was limited by the popularity
of serotherapy [5 11. Since the pneumococcus did not exhibit
the same extreme susceptibility to sulfanilamide as did Srrep-
tococcus pyogenes, Whitby [88] undertook a systematic
search for a related chemical compound with good in vitro
activity but relatively low toxicity. According to this author,
"these experiments represent the one striking success in the
chemotherapy of pneumococcal infections in an assessment
of no less than 64 related sulfanilamide compounds." One of
these 64 derivatives possessed the proper combination of low
toxicity and good in vitro activity; this compound was re-
ferred to as 2-(p-aminobenzenesulfonamido) pyridine, or
simply sulfapyridine. (Whitby's approach has been the basis,
in more modem times, for selection of a particular formula-
tion of a given antimicrobial compound for further clinical
testing.) This work [88] was followed only 5 weeks later in
The Lancer by the study of Evans and Gaisford [89], who
reported that treatment with sulfapyridine reduced the over-
all case-fatality rate from 27% to 8% among patients with
pneumonia (including 100 with lobar pneumonia) at the
Dudley Road Hospital in Birmingham, England.

Thus, for a brief period, sulfapyridine appeared to be the
treatment of choice for pneumococcal infections. By 1943,
however, in an early example of an increasingly important
problem, sulfonamide-resistant strains of S. pneumoniae
were reported by Tillett et al. [90].

Penicillin.  In 1929 Fleming [9 l] discovered the antibac-
terial properties of the fungus-derived substance that came to
be called penicillin. The third subject to receive this drug (by
topical application)-and the first to show any clinical bene-
fit-was suffering from pneumococcal conjunctivitis [5 11.
Compared with sulfanilamide, penicillin possessed a number

of superior attributes, including greater potency per unit,
minimal influence of inoculum size on effectiveness, and
lack of interference by the breakdown products of protein
hydrolysis [92]. However, the efficacy of readily synthesized
sulfanilamide in treating pneumococcal infections, coupled
with the difficulty of obtaining sufficient quantities of peni-
cillin, meant that the full potential of the latter drug in com-
bating the pneumococcus was not immediately realized.

In 1939 Dubos [93] discovered the first naturally occur-
ring antimicrobial compound with demonstrable activity in
vitro against a bacterial pathogen. This compound was
named gramicidin, and the activity demonstrated was
against S. pneumoniae. Unfortunately, like optochin, gramici-
din proved to be toxic in mice [94] and dogs [95], and this
toxicity effectively ruled out its use in humans. On the posi-
tive side, however, the identification of this compound by
Dubos did prompt Chain and colleagues [96] to reevaluate
the antibacterial properties of penicillin in 1940. This reanal-
ysis was made possible by methods that these investigators
developed at Oxford for the isolation of penicillin in large
quantities and for the rapid assay of its inhibitory power, as
described in detail by Abraham et al. [92] in a landmark
paper appearing in The Lance1 in 194 1. In the same elegant
paper, this group detailed their dramatic results in the treat-
ment of life-threatening infections caused by gram-positive
cocci, including S. pneumoniae. As a result, the approach to
the treatment of pneumococcal infections was changed for-
ever. In I943 Keefer et al. [97] reported a series of 500 cases
in which penicillin was used with great success in the treat-
ment of a variety of staphylococcal and streptococcal (in-
cluding pneumococcal) infections, mostly those resistant to
sulfonamides. According to a personal communication from
Louis Weinstein:

Having obtained small amounts of penicillin through his con-
tacts with researchers at Oxford, Dr. Chester Keefer first tried
to treat patients at the Boston Memorial Hospital with 5,000
units of penicillin every four hours for viridans streptococcus
endocarditis. When that treatment failed, Keefer (the supervi-
sor) and [I] (the acting intern) turned to the treatment ofpneu-
mococcal pneumonia with dramatic results. This was the first
disease for which penicillin was used successfully in the
United States.

The next year, Tillett et al. [98] reported on the use of
penicillin in 46 cases of pneumococcal pneumonia and 8
cases of pneumococcal empyema, again with excellent re-
sults. This study was useful in further defining proper treat-
ment for pneumococcal infections, since the Keefer
study-conducted during a period of great dedication to the
U.S. effort in World War II-was focused narrowly "toward
those infections that are most likely to occur in our armed
forces." These investigations yielded convincing evidence of
the value of penicillin in the treatment of a variety of bacte-
rial infections. As Mufson has pointed out [99], studies of


Watson et al.

CID 1993; 17 (November)

918

100 d

60 -

20-

0' I       I
   12-29    30.49    50-69    70+
          Age Group

Figure 2. Graphic representation of data taken from Mufson
[99], showing a uniform decline of 40%-50% in mortality asso-
ciated with pneumococcal bacteremia for every age group after the
introduction of penicillin for the treatment of pneumococcal infec-
tion. Data for mortality due to pneumococcal bacteremia in the era
preceding the introduction of penicillin were obtained at the Bos-
ton City Hospital, while those for mortality after the introduction
of this drug were obtained at Kings County Hospital in Brooklyn,
New York ( 1952- 1962) and at Cook County Hospital in Chicago
( 1967- 1970).

mortality from serious pneumococcal infection as a function
of age showed a dramatic reduction after the introduction of
penicillin (figure 2).

Studies ofthe pneumococcus were among the first to docu-
ment the clinical relevance of penicillin-binding proteins
(PBPs) in the development of resistance to penicillin. Close
examination of the data from the paper published in The
Lancet in 1941 by Abraham et al. reveals that even the first
studies of the in vitro susceptibility of S. pneumoniae to peni-
cillin detected a biphasic pattern. Specifically, one group of
pneumococci was at least 30 times as susceptible to the drug
as was the other, yet both groups included isolates of the
same serotypes. In fact, serotype 19F, recently associated
with both moderate and high-level resistance to penicillin,
was originally identified by Abraham et al. [92] as being the
serotype of one of the less sensitive isolates. Although to our
knowledge the PBP profiles of these strains have never been
examined, it is likely that differences in PBPs were responsi-
ble for the discrepancy. By 1943 it had been shown that
pneumococcal resistance to penicillin could be induced in
vitro [ 1001 or in vivo (in the mouse) [ 1011. In light of these
findings, the reports by Hansman and Bullen [ 1021 of a
highly penicillin-resistant pneumococcus and later by Ap-
pelbaum et al. [ 103, IO41 and by Jacobs et al. [ 1051 of a large
outbreak of penicillin-resistant pneumococcal infections are
surprising, not so much because penicillin-resistant clinical
isolates of S. pneumoniae were identified but because such
isolates took so many years to appear.

The mechanism by which resistance to penicillin arises in
pneumococci has been shown to be decreased binding of the
drug to PBPs, which are also known as transmembrane car-
boxypeptidases-enzymes involved in cell wall synthesis.

(See Waxman and Strominger [ 1061 for a review.) The con-
cept of such surface proteins was developed by Spratt [ 1071
in studies with Escherichia coli; pioneering investigations of
decreased penicillin binding to pneumococci were published
in 1954 by Eagle [ 108, 1091, albeit without knowledge of
specific surface-associated PBPs. The large-scale outbreak of
penicillin-resistant pneumococci in South Africa discussed
above [ 1051 led to the identification of one of the first clini-
cal correlates of the PBP concept-namely, that alteration of
these proteins contributed to the development of resistance
to penicillin. In 1980 Hakenbeck et al. [ 1 IO] reported alter-
ations in the PBPs of clinical isolates of pneumococci asso-
ciated with increased resistance to penicillin. Zighelboim
and Tomasz [ 1 I I] extended this finding to penicillin-resis-
tant isolates from South Africa and further described the
mechanism of resistance. Additional studies of PBPs have
been reported by Hakenbeck et al. [ 112, I 131, Chalkley and
Koornhof [ 1141, Dowson and colleagues [ 1151, and Jabes et
al. [ 1161. Moreover, in two of only a few well-documented
instances, pneumococci have been shown to be both the do-
nors of altered PBP DNA sequences to other streptococcal
species [ 1171 and the recipients of such sequences from an-
other streptococcal species [ 1181. Horizontal transfer of PBP
genes has also been demonstrated among natural popula-
tions of pneumococci [ 119, 1201, and penicillin-resistant
clones have even been shown to have taken transcontinental
journeys [ 12 I]. The increasing frequency of penicillin-resis-
tant pneumococci [ 122- 1241 is especially worrisome in light
of the trend toward higher levels of resistance to vancomycin
among enterococci, since horizontal genetic transfer of the
latter resistance to pneumococci seems likely.

Discovery of the Transforming Principle

In 19 16 Stryker [ 1251 described changes that occurred in
pneumococci upon growth in broth containing homologous    '

immune serum. She noted that, when virulent strains were
cultured in this fashion, they became less virulent, produced
less capsular material, were more readily ingested by phago-
cytes, and displayed altered antigenic properties. Griffith
built on these data [126], borrowing the terminology of
Arkwright [ 1271 to describe the appearance of colonies of
dysentery bacilli on plates containing homologous immune
antiserum. Smooth ("S") colonies, as defined by Griffith
[ 1281, possess a lustrous, mucoid, macroscopically apparent
colonial phenotype attributable to the presence of a polysac-
charide capsule; agglutinate in the presence of homologous
antisera; cause fatal infections in laboratory animals; and,
when injected into rabbits, stimulate the production of pro-
tective antibodies. Rough ("R") forms do not possess the
extracellular polysaccharide capsule; are avirulent; and,
when injected into rabbits, lead to the production of antisera
specifically reactive only with other rough pneumococci.

Griffith showed that some induced rough forms could re-


CID 1993; I7 (November)

The Pneumococcus in Biomedical Research

919

vert to the smooth form in vivo, while others could not. Even
rough forms that never spontaneously reverted to capsule
production, which he regarded as completely "dissociated"
(i.e., unable to produce a capsule), could be transformed
back to their original capsular types by a novel technique
that Griffith himself pioneered. This procedure involved the
concomitant injection into mice of heat-killed smooth pneu-
mococci of the same or a different capsular type together
with the nonrevertible rough strain. Under these conditions
Griffith found that the rough form not only could be made to
revert to its original capsular type but also could acquire the
capsular type of the heat-killed organism. He did not under-
stand the significance of this finding at the time, but his re-
sults were quickly verified by Neufeld and Levinthal [ 1291.
Dawson and colleagues [130-l 331 and Alloway [ 134, 1351
appreciably extended these observations. Alloway demon-
strated the phenomenon in vitro, using extracts of S. pneu-
moniae; in his first study he dissolved the bacteria by re-
peated freezing and thawing, while in the second he added
sodium deoxycholate to bacterial suspensions for lysis. Tra-
gically, the scientific careers of both Griffith and Neufeld-
the former British and the latter German-were cut short by
incidents directly related to World War II [ 1361.

It was not until 1944 that a landmark (and at that time
controversial [ 1361) paper was published by Avery,
MacLeod, and McCarty [ 1371. Their studies showed conclu-
sively that DNA-and not some other molecule-consti-
tuted the genetic material responsible for phenotypic
changes during transformation. In a now-famous letter to his
brother Roy [ 1381, Oswald Avery was cautiously optimistic:

. [A]t last perhaps we have it. . . [T]his [fibrous] sub-
stance is highly reactive and on elementary analysis conforms
very closely to the theoretical values of pure desoxyribose nu-
cleic acid (thymus) type (who could have guessed it).... If we
are right, and of course that is not yet proven, then it means
that nucleic acids are not merely structurally important but
functionally active substances in determining the biochemical
activities and specific characteristics ofcells and that by means
ofa known chemical substance it is possible to produce predict-
able and hereditary changes in cells. This is something that has
long been the dream of geneticists.

This observation later was strongly supported in two ways.
First, after publication of the 1944 paper, McCarty, while
still working in Avery's laboratory, showed that "treatment
of the transforming principle with concentrations of DNase
(a partially purified pancreatic enzyme which is capable of
depolymerizing DNA) so small that only a slight fall in vis-
cosity (of the DNA solution) occurs causes a marked loss of
biological activity" [ 1391. This finding constituted further
proof of the nucleic acid nature of the transforming princi-
ple. In a brilliant foreshadowing of the future work on DNA
by Watson and Crick, McCarty stated: "It remains one of the
challenging problems for future research to determine what

sort of configurational or structural differences can be dem-
onstrated between desoxyribonucleates of separate specilici-
ties" [ 1391. Later, Hotchkiss [ 1401 showed that, in addition
to the genes encoding capsule production, those sequences
specifying resistance to the powerful antibiotic penicillin
could be transferred to a previously penicillin-sensitive
pneumococcus by DNA isolated from a penicillin-resistant
pneumococcus. Although Hotchkiss joined Avery's group in
1935, he did not become involved in the work on S. pneu-
moniae transformation work until 1946, when (as recalled in
an unpublished speech by Dr. Maclyn McCarty nominating
Dr. Hotchkiss for an honorary doctorate in humane letters at
the Rockefeller University in 1988) he

quickly made a number of advances that clarified the trans-
forming reaction and addressed the criticism that the apparent
activity of the transforming DNA must be due to contaminat-
ing protein (as suggested by Alfred Mirsky).... [Hotchkiss] an-
swered the challenge ofcontaminating protein by further puri-
fication of the pneumococcal DNA without loss of activity
until only minute traces of protein remained. In other experi-
ments initiated at this time, he broadened the genetic implica-
tions of transformation by showing that traits other than cap-
sule formation (e.g., antibiotic resistance) could be introduced
by the transfer of DNA. As a result, all but the most
hardened skeptics were convinced that DNA is the bearer of
genetic information.

In this instance, possibly as never before, the pneumococ-
cus was at center stage in a critically important scientific
discovery; one that in fact initiated the era of molecular biol-
ogy and is arguably one of the single greatest achievements in
biological science in the twentieth century. It is known that
Hershey and Chase, who performed the classic experiment
showing that infecting bacteriophages inject only DNA into
their bacterial targets (an event that results in the production
of progeny phages), were inspired by their knowledge of
Avery's paper [48]. Moreover, Watson stated in two different
passages of The Double Helix. the best-selling account of the
discovery of the structure of DNA, that both he (through his
mentor, Salvatore Luria) and Francis Crick became con-
vinced that DNA was the genetic material by reading the
paper by Avery et al. [ 1411.

While Avery and associates showed that the transforming
principle actually encoding the encapsulation phenotype of
S. pneumoniae consisted exclusively of DNA, the genes re-
sponsible for capsule production in the pneumococcus have
never been cloned. In 1959 Austrian and colleagues [ 142,
1431 showed that DNAs that were obtained from unencapsu-
lated derivatives of two pneumococcal serotypes and that
contained separate mutations in a common biosynthetic
pathway could complement each other and produce "binary
capsulation." The most important conclusion to be drawn
from this work was that "the capsular genome appears to
have a specific location in the total genome of the cell, this
location being occupied by the capsular genome of whatever


920

Watson et al.

CID 1993; I7 (November)

capsular type is expressed by the cell... [and] the new capsu-
lar genome is transferred to the transformed cell as a single
particle of DNA" [ 1431. R avin [ 1441 demonstrated the same
concept in the same year-namely, that DNA involved in
encapsulation consists of a discrete contiguous unit, or cas-
sette. Coffey et al. [ 1201 have recently presented indirect
evidence for horizontal transfer of encapsulation genes to a
penicillin-resistant pneumococcus in nature. The genetics of
the encapsulation piocess has been investigated in a few
other bacterial species, with E. coli K12 [ 145-1471 and H.
inJluenzae type b [ 148- 15 l] most extensively studied. A se-
quence putatively involved in encapsulation of S. pneumon-
iae serotype 3 has recently been targeted [ 1521 and cloned in
our laboratory [ 1531 and is currently the subject of intensive
DNA sequencing efforts. Preliminary data suggest the pres-
ence ofat least two loci: one unique to each serotype and one
common to all serotypes.

Proteins as Virulence Factors

In recent years considerable effort has been directed to the
question of whether accessory proteinaceous virulence fac-
tors exist in the pneumococcus, as they do in many bacteria,
including some streptococci. Boulnois [ 1541 has extensively
reviewed a number of putative proteinaceous virulence fac-
tors of the pneumococcus, two of which deserve mention
here since they have been the subject of much recent work.
The first is the sulfhydryl-activated but nonsecreted hemoly-
sin referred to as pneumolysin [ 1541. During the 1980s a
number of studies (see [ 1541 for references) convincingly
showed that pneumolysin alone can produce all the manifes-
tations of pneumococcal pneumonia. Since pneumolysin is
liberated upon autolysis of pneumococci, it is not difficult to
visualize a role for this toxin in disease. Pneumolysin may, in
fact, eventually be shown to be the elusive toxin long consid-
ered a major contributor to the morbidity and mortality asso-
ciated with pneumococcal pneumonia. It remains to be seen,
however, whether the amount of toxin produced per bacte-
rial cell varies among strains. Such variation could begin to
explain observed differences in virulence among strains of
the same serotype ([ 1551 and authors' unpublished observa-
tions). The surface-associated protein pspA [ 156, 1571 may
also serve a still-unidentified function in virulence, given
that isogenic pspA strains of some (but not all) serotypes
examined to date exhibit greatly reduced virulence [ 1581.

Future Trends in Pneumococcal Research

Predicting the future is not a science, even when science is
the subject under discussion. However, the study of what
Avery called the sugar-coated microbe has yielded a number
of unexpected and profoundly important basic biological dis-
coveries (as outlined herein), and we are convinced that the
jigsaw puzzle of pneumococcal pathogenesis will continue to

attract investigators whose efforts will yield results with
broad implications.

Research on improved polysaccharide-protein conjugate
vaccines will continue to be an area of great interest over the
next several years, since a vaccine that is efficacious in very
young children and other high-risk groups remains a high
priority. Elucidation of the molecular basis for capsule pro-
duction among pneumococci is a field in which we and other
researchers are presently quite involved. The recent identifi-
cation of short (154-base-pair) repeated-sequence elements
strategically located with respect to virulence genes and to
metabolically important genes in the S. pneumoniae genome
[ 1591 immediately suggested to their discoverers the possibil-
ity of coordinated regulation of important genes by these
elements. Elucidation of the molecular machinery of such a
control mechanism could add much to our understanding of
pneumococcal pathogenesis.

The trend toward an increased incidence of penicillin-re-
sistant pneumococci shows no signs of reversing and is partic-
ularly alarming in some locations. As has been discussed,
resistant clones in the nasopharyngeal cavities of colonized
travelers are probably being disseminated from continent to
continent. If resistance to vancomycin can be passed from
enterococci to pneumococci via horizontal gene transfer, we
may soon see multidrug-resistant pneumococcal infections
that are virtually untreatable. New antibiotics and different
therapeutic strategies obviously need to be developed. Con-
current administration of new types of nonsteroidal anti-in-
flammatory drugs and antibiotics may be promising for the
treatment of pneumococcal meningitis.
Given this wealth of possibilities, the future of pneumo-
coccal research promises to be at least as exciting as its past.

Acknowledgments

The authors thank Drs. Rollin Hotchkiss and Vincent Fis-
chetti for their suggestions and insights concerning research on
the transforming principle; Dr. Maclyn McCarty for access to an
unpublished speech and for encouragement of his younger col-
leagues in the study of the pneumococcus; and Dr. Maurice
Mufson for permission to adapt his tabulated data into a figure.

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