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Competition among Streptococcus pneumoniae for intranasal colonization in a mouse model
Vaccine 18 (2000) 2895±2901
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Competition among Streptococcus pneumoniae for intranasal colonization in a mouse model M. Lipsitch a, b,*, J.K. Dykes a, S.E. Johnson a, E.W. Ades a, J. King c, D.E. Briles c, G.M. Carlone a a
Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA b Department of Biology, Emory University, Atlanta, GA 30322, USA c Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Received 29 November 1999; accepted 24 January 2000
Abstract Widespread use of conjugate vaccines against Streptococcus pneumoniae, by reducing carriage of S. pneumoniae serotypes included in the vaccine, may result in an increase in nasopharyngeal carriage of Ð and disease from Ð nonvaccine serotypes of the same species. Mathematical models predict that the extent of such replacement will depend positively on the degree to which carriage of vaccine-type S. pneumoniae inhibits acquisition of nonvaccine-type pneumococci, and may depend negatively on the inhibition of vaccine-type pneumococci by nonvaccine-type pneumococci. We used a mouse model of intranasal carriage of pneumococci to test whether such inhibition occurs between dierent pneumococcal strains. Mice carrying a streptomycinresistant derivative of S. pneumoniae BG9163 (serotype 6B) as a resident strain showed reduced levels of colonization when challenged intranasally by optochin-resistant derivatives of the same strain and of a serotype 23F pneumococcus, BG8826. Inhibition could be overcome by increasing the dose of the challenge strain. Carriage of optochin-resistant BG9163 did not inhibit acquisition of the streptomycin-resistant variant. Colonization by a challenge strain did not signi®cantly aect the level of colonization with the resident strain. These results provide evidence that is consistent with several hitherto untested assumptions of mathematical models of serotype replacement and suggest that a biological mechanism exists that could account for serotype replacement that is observed in clinical trials. The ®ndings provide a basis for further studies of in vivo interactions between strains of S. pneumoniae. Published by Elsevier Science Ltd. Keywords: Streptococcus pneumoniae; Serotype replacement; Nasopharyngeal carriage; Mathematical models; In vivo models
1. Introduction and background Conjugate vaccines against Streptococcus pneumoniae are currently in clinical trials [1±5]. These vaccines immunize against 7, 9, of the 90 known pneumococcal serotypes. Preliminary results from these ongoing trials in infants indicate that these vaccines are highly pro* Corresponding author: Department of Epidemiology, Harvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA. Tel.: +001-617-432-4559; fax: +001-617-566-7805. E-mail address: [email protected] (M. Lipsitch). 0264-410X/00/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 0 4 6 - 3
tective against invasive disease [1] and oer signi®cant protection against pneumonia [1] and otitis media [1,4], from the serotypes included in the vaccine. In addition, the vaccine oers partial protection against nasopharyngeal carriage of the included serotypes [5± 7]. Because nasopharyngeal carriers of pneumococci are the major source for transmission of these bacteria, conjugate vaccines are expected to reduce transmission and prevalence of vaccine-type pneumococci. This reduction in pneumococcal transmission should help to reduce the incidence of disease from vaccine-type pneumococci among unvaccinated individuals in communities where vaccination is used, via herd immunity
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(indirect eects), a hypothesis that is currently being tested in community-randomized clinical trials (K. O'Brien, personal communication). Vaccine-induced reductions in the prevalence of vaccine-type pneumococci may also have the unintended eect of increasing the prevalence of those pneumococcal serotypes not included in the vaccine. This phenomenon of ``serotype replacement'' would be expected to occur if carriage of vaccine-type pneumococci reduces a host's probability or intensity of colonization by nonvaccine-type pneumococci, via competition between bacteria types to colonize the nasopharynx. Preliminary results from clinical trials have suggested that serotype replacement occurs at least at the level of nasopharyngeal carriage [5±7]. Thus far, no increase in disease from nonvaccine-type pneumococci has been observed in the one trial where it has been measured [1]; however, these optimistic results should be tempered by the expectation that serotype replacement may be more pronounced following widespread use of a vaccine in a community than in a clinical trial [8]. Mathematical models are valuable tools for assessing the population-wide consequences of public health interventions such as the introduction of vaccines [9], and in particular for assessing the potential for serotype replacement following vaccination against the pneumococcus [8,10]. Such models may be used to predict, for instance, which biological parameters determine the extent of serotype replacement following vaccination, and how such replacement can best be measured during clinical trials [8,10]. For example, a simple mathematical model of the interaction between
Fig. 1. Predictions of a mathematical model [10] for the maximum possible serotype replacement expected following mass vaccination with a conjugate vaccine. Maximum replacement is de®ned as the absolute increase in prevalence of nonvaccine-type pneumococcal carriage that would be expected if vaccine-type carriage were eradicated. rN (shown on the x-axis) measures the inhibition of nonvaccine-type pneumococcal colonization by vaccine-type carriage; rV measures the inhibition of vaccine-type by nonvaccine-type. The potential for replacement is greatest when nonvaccine-type colonization is strongly reduced by vaccine-type carriage (high rN ) and when vaccine-type colonization is not strongly reduced by nonvaccine-type carriage (low rV ). Parameters for this ®gure: R01=1.8; R02=2.2; c1=1ÿrV; c2=1ÿrN.
vaccine-type and nonvaccine-type pneumococci [10] predicts that the extent of serotype replacement anticipated in a population with high vaccine coverage depends strongly on the ability of vaccine-type pneumococci, carried in the nasopharynx, to reduce the incidence of colonization with nonvaccine-type pneumococci. It also depends, albeit less strongly, on the opposite interaction: the extent to which carriage of nonvaccine-type pneumococci reduces the incidence of colonization with vaccine-type pneumococci. This relationship, as predicted by the mathematical model described in [10], is shown in Fig. 1. The maximum extent of serotype replacement, measured as the absolute increase in the prevalence of carriage of nonvaccine-type pneumococci in a population where vaccination has eliminated carriage of vaccine-type pneumococci, is shown on the vertical axis. The variable on the horizontal axis, rN, measures the strength of competition between a resident, vaccine-type strain and a potentially colonizing nonvaccine-type strain. Speci®cally, rN is de®ned as the reduction in incidence of new colonization by nonvaccine-type S. pneumoniae in individuals currently carrying vaccine-type S. pneumoniae. The dierent curves refer to dierent levels of competition in the opposite direction: rV is de®ned as the reduction in incidence of new colonization by vaccine-type S. pneumoniae in individuals currently carrying nonvaccine-type S. pneumoniae. As Fig. 1 shows, the potential for serotype replacement is greatest if carriage of vaccine-type strongly inhibits acquisition of nonvaccine-type, and is somewhat reduced if carriage of nonvaccine-type strongly inhibits acquisition of vaccine-type. The parameters used in Fig. 1 are arbitrary, but the relationship shown occurs for a broad range of parameter choices. The detailed predictions of the mathematical model have been described previously [8,10]. Several of the model's assumptions, although consistent with present knowledge of the biology and epidemiology of the pneumococcus, have not been formally tested. For example, although there are plausible mechanisms by which dierent strains of pneumococci might compete for colonization of the nasopharynx [11], there are no direct measurements of this phenomenon. As a result, it is not known whether carriage of one pneumococcal strain reduces the probability of acquiring a second strain, or whether it (instead or in addition) reduces the population size attained by a second strain that does successfully colonize a host. To take another example, the model assumes that the population of one strain, once established in the nasopharynx, is not aected by acquisition of a second strain by the same host. In this report, we describe an experimental system in which these assumptions of the model can be tested in vivo. Results are presented to show that, in a mouse
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model, intranasal carriage of one pneumococcal strain can inhibit acquisition of a second pneumococcal strain; that the existence and strength of this inhibition, while repeatable under the same experimental conditions, depends on the strains chosen and the doses administered; and that the inhibition can take the form of reduced probability of colonization and/or reduced bacterial counts in animals that are colonized. Furthermore, in these experiments acquisition of a second strain does not aect the levels of colonization by a resident strain. Thus, we demonstrate that dierent strains of pneumococci compete for intranasal colonization in mice, a mechanism that could result in serotype replacement following widespread use of conjugate vaccines. We provide an experimental system in which these interactions can be assessed systematically for a larger number of strains. 2. Methods 2.1. Bacterial strains S. pneumoniae strains were antibiotic-resistant mutants isolated from strains BG9163 (serotype 6B) and BG8826 (23F) [12]. Optochin-resistant (Opt1) mutants were isolated on tryptic soy agar + 5% sheep's blood plates (SBA) containing 5 mg/l optochin, and streptomycin-resistant (Str1) mutants were isolated on SBA plates containing 200 mg/l streptomycin. Isolates were restreaked on antibiotic plates and then grown to late log phase and plated on both antibioticcontaining and antibiotic-free plates to con®rm the stability of the resistance markers. For use in inoculations, bacteria were grown to optical densities of approx. 0.4 (492 nm) in Todd±Hewitt Broth supplemented with 0.5% yeast extract (THY), and frozen at ÿ708C in 1 ml aliquots in THY+10% glycerol. At the time of inoculation, aliquots were thawed, centrifuged and resuspended in physiologic saline (Gibco) twice, and diluted to the desired density, which was con®rmed by plating. 2.2. Mouse inoculation and sampling Female C57BL/6J (Jackson Laboratories, Bar Harbor, ME, USA) mice aged 5±7 weeks at the beginning of the experiment were used in all experiments. Committee approval was obtained at the Centers for Disease Control and Prevention for animal care and use. Mice were inoculated with de®ned numbers of colonyforming-units (cfu; speci®c numbers given in the Results section) intranasally by pipetting 10 ml of saline containing the bacteria onto the nares [12]. For sampling, mice were anesthetized by an intraperitoneal injection of 0.02 ml ketamine and 0.01 ml xylazine
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diluted in 0.9% NaCl, and sacri®ced by CO2 inhalation. Nasal washes were obtained by washing approx. 0.25 ml of saline through the trachea and collecting the ®rst 0.1 ml of nasal wash as it exited the nares, as described [12]. The wash was collected in sterile tubes already containing 0.1 ml of saline, and stored on ice until diluted and plated on selective media (SBA plates supplemented with 2.5 mg/l gentamicin to suppress growth of organisms other than S. pneumoniae, plus either streptomycin or optochin as above). On occasion (at most one mouse per group) we failed to obtain any nasal wash; this accounts for variations in numbers between groups in the same experiment. 2.3. Experimental protocol For basic competition experiments, groups of 10±12 mice were inoculated intranasally on day 0 with a resident strain or mock-inoculated with saline. On day 3, both groups were challenged with a challenge strain carrying a dierent antibiotic resistance marker. On day 7, mice were sacri®ced and nasal washes obtained and plated on antibiotic selective plates to dierentiate the challenge strain from the resident strain. Competition was de®ned as a reduction in the number of mice colonized with the challenge strain in animals carrying the resident strain vs. controls. Numbers of cfu recovered were compared and statistical signi®cance tested with the two-tailed Mann±Whitney U-test (all mice in which no challenge strain was recovered were considered ties for the lowest rank). Dierences in the number of mice colonized (disregarding level of colonization) were assessed using Fisher's exact test. As noted individually below, the competition experiment was also performed over a 14-day period, with inoculations on days 0 and 7 (rather than 0 and 3) and sacri®ce on day 14. To assess the eect of a challenge strain on the resident strain, the protocol was reversed. On day 0, all mice in two groups were given a resident strain intranasally. On day 7, one group was given saline, the other a challenge strain. On day 14, the mice were sacri®ced, and nasal washes obtained and plated. Competition was de®ned as a reduction in the carriage of resident strain by mice given the challenge strain vs. controls given saline. 3. Results 3.1. Carriage of a resident strain could inhibit colonization by a challenge strain Fig. 2 shows the results of 7-day [Fig. 2(a)] and 14day [Fig. 2(b)] experiments in which BG9163Str1 was the resident strain and the otherwise isogenic
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BG9163Opt1 was the challenge strain. Inocula were 7 104 cfu (resident strain) and 4 104 cfu (challenge strain) for the 7-day experiment, and 6 104 cfu (resident strain) and 4 104 cfu (challenge strain) for the 14-day experiment. Competition was observed both in the 7 and 14-day experiment. In the 7-day experiment, carriage of the resident strain BG9163Str1 reduced the level of carriage of the challenge strain BG9163Opt1 by about 25-fold (median cfu recovered 102.9 vs. 104.3, p < 0.005), with 10/10 mice colonized in each group. In the 14-day experiment, the eect was larger. Challenge strain was recovered from 5/10 mice carrying the resident strain, with a median of 101.4 cfu in the mice carrying any recoverable challenge strain; by contrast, 9/10 control mice were colonized with a median recovery of 104.0 cfu/nasal wash in the colonized mice. The dierence in levels of colonization (with 0 colonization assessed as ties) was highly signi®cant, p < 0.0005 (Mann±Whitney U-test). The dierence in the proportion of mice colonized did not reach statistical signi®cance ( p = 0.15, Fisher's exact test). Similar results were obtained when the challenge strain was BG8826Opt1 (see Fig. 3 below) at approximately the same dose. 3.2. Inhibition was not reciprocal The 14-day experiment described above was repeated with the strains in opposite roles: BG9163Opt1 was the resident strain and BG9163Str1 was the challenge strain. No signi®cant dierence in carriage of the challenge strain between carriers of the resident strain (8/9 mice colonized, median of colonized mice = 104.0 cfu, range 103.6±104.6 cfu) and con-
trols (9/9 mice colonized median = 104.3 cfu, range 103.6±104.6 cfu) was detected. 3.3. The inhibitory eect of a resident strain on acquisition of a challenge strain depended on the dose of the challenge strain Fig. 3 shows the results of 7-day experiments in which BG9163Str1 (inoculum: 8 105 cfu) was the resident strain and BG8826Opt1 was the challenge strain. Note that in contrast to Fig. 2, this experiment used unrelated strains of dierent serotypes, and several doses of the challenge strain were administered, with inocula of 5 104, 5 105 and 5 106 cfu, respectively. Carriage of the resident strain signi®cantly inhibited acquisition of the challenge strain at the lowest challenge dose. At this dose, 7/12 mice carrying the resident strain acquired the challenge strain, with a median of 102.1 cfu among those colonized; 10/11 control mice acquired the challenge strain, with a median of 102.7 cfu among those colonized, p < 0.01 (Mann± Whitney U-test). However, the inhibition did not appear at higher doses of the challenge strain. 3.4. Inoculation of a challenge strain had little or no eect on carriage of a resident strain Fig. 4 shows the results of reciprocal 14-day experiments in which mice were given a resident strain (either BG9163Opt1 or the otherwise isogenic BG9163Str1) at day 0, challenged with the opposite strain or mock-challenged with saline at day 7, and sacri®ced and sampled at day 14. As the ®gures show, there was a small (two to four fold) reduction in the median level of the resident strain in mice that had
Fig. 2. Inhibition of colonization by a challenge strain in mice already carrying a resident strain. Carriage of the resident strain, BG9163Str1 inhibited acquisition of the challenge strain BG9163Opt1 in 7-day (a) and 14-day (b) experiments. Shown are bacterial counts of the challenge strain (®gures give medians of colonized animals) in controls and carriers of the resident strain. N = 10 mice per group. Italic ®gures at the bottom (``NONE'') designate mice from which no challenge strain pneumococci were recovered. P values refer to Mann±Whitney U-test comparing counts of the challenge strain in controls to counts in carriers.
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Fig. 3. Relationship between eect of resident strain (BG9163Str1) on acquisition of a challenge strain (BG8826Opt1) and the dose of the challenge strain. Acquisition of the challenge strain was inhibited in mice carrying the resident strain when the dose of the challenge strain was low, but at higher doses the challenge strain was able to colonize resident strain-carrying mice with equal success to that achieved in controls. N = 12 mice per group except for carriers in 5 104 and 5 106 groups, for which N = 11. Italic ®gures at the bottom (``NONE'') designate mice from which no challenge strain pneumococci were recovered.
been challenged with the challenge strain, but the variation within groups was substantial, and the reduction was not statistically signi®cant. 4. Discussion The experiments reported here demonstrate that S. pneumoniae carried intranasally can interfere with nasal colonization by a second strain of S. pneumoniae to which the animal is exposed. The removal of this interference by reductions in carriage of vaccine-type S. pneumoniae provides a biological mechanism that could lead to serotype replacement following the introduction of a conjugate pneumococcal vaccine into a population. As described in Section 1, mathematical models pre-
dict that the extent of the increase in carriage of nonvaccine-type pneumococci expected following introduction of a vaccine depends on many factors, including the strength of the competitive interaction between pneumococci. These experiments on a few strains demonstrate that several of the basic assumptions of existing mathematical models are correct: (a) carriage of one strain can reduce the level of colonization by a second strain; (b) once established in the nasopharynx, carriage of a resident strain is not aected signi®cantly by acquisition of a second strain; (c) dierent pneumococci vary in their competitive abilities. Another observation that emerges from this study is that antibiotic resistance markers appear to in¯uence components of the ®tness of pneumococci in vivo, where ®tness is de®ned as the ability of a de®ned
Fig. 4. Recovery of the resident strain in control mice vs. mice colonized with a challenge strain, in experiments lasting 14 days. Both strains were antibiotic resistant mutants of BG9163: (a) resident strain: Str1, challenge strain Opt1; (b) resident strain: Opt1, challenge strain Str1. A slight dierence was observed in each case, but neither was statistically signi®cant. N = 10 mice per group. Inocula: 6 104 cfu of Str1 resident, 4 104 cfu of Opt1 challenge; 3 104 cfu of Opt1 resident, 7 104 cfu of Str1 challenge. N = 10 mice per group.
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inoculum to colonize a mouse and grow to high population sizes. This is most obvious from the experiments with otherwise isogenic isolates of BG9163 described in Sections 3.1 and 3.2. In these experiments, we observed that the streptomycin-resistant variant was able to inhibit the acquisition of the optochin-resistant, streptomycin-sensitive variant, but the opposite did not occur. This observation is consistent with previous observations in pneumococci [13,14] and other bacterial species [15], in which antibiotic resistance has resulted in a reduction in bacterial growth rate, virulence and/or competitive ability in vitro and in vivo. One consequence of this observation is that competition studies of S. pneumoniae must be designed in such a way that interpretation of the data does not depend on the assumption that antibiotic resistance markers have no eect on bacterial ®tness. In the present study, this is accomplished by measuring competition as a dierence in the performance of one strain (with the same antibiotic resistance marker) under dierent conditions, for example, comparing colonization with BG9163Opt1 in the presence or absence of BG9163Str1. The experiments described here demonstrate that interference between pneumococci can occur in vivo. These preliminary studies nonetheless have several limitations, most of which can be addressed by further experimental work with a wider range of strains. First, the numbers of mice involved was relatively small, and would not have had the power to detect subtle eects of challenge strains on resident strains. However, the remarkable concordance between the levels of resident strain in mice that were, and were not, challenged with a second strain suggests that such eects, if they occurred, were not large. Second, the small numbers prevented us from determining whether carriage of a resident strain signi®cantly reduced an animal's probability of acquiring a challenge strain, although we were able to show that it reduced the level of colonization (which sometimes took the form of preventing colonization altogether). Third, and perhaps most importantly, the experiments were limited to three strains, which were derived by isolation of antibiotic resistant mutants of two human isolates which were both of serotypes included in the vaccine. Ideally, one would like to use a wider variety of primary human isolates without introduced antibiotic resistance mutations; these studies are currently underway. To use the mathematical model to make more speci®c predictions about the potential for serotype replacement, it will be necessary to measure competitive interactions in a wider range of strains, encompassing a number of vaccine-serotype and nonvaccineserotype pneumococci. For example, if one found that vaccine-type pneumococci were generally better competitors for nasopharyngeal colonization than nonvac-
cine-type pneumococci (meaning that vaccine-type strongly inhibit acquisition of nonvaccine-type but are only weakly inhibited by nonvaccine-type), then the potential for serotype replacement would be relatively high, as suggested by Fig. 1. The experimental techniques described here provide a straightforward way to assess these parameters for any pair of strains, subject of course to the caveats attending any animal model. In addition to investigating the range of interactions among various natural pneumococcal isolates within an individual, the experimental system described here provides the opportunity to address a number of other questions. One such question is the nature of the mechanism(s) of inhibition between strains. This question is not only of biological interest, but also has important consequences for serotype replacement. We have shown that pneumococci compete directly within a host, and the mathematical models indicate that this can lead to serotype replacement when transmission of vaccine serotypes is reduced. If carriage has existed for several weeks, or if the host is already primed to the bacterial antigens, it is possible that speci®c antibodies could be elicited which could have an eect on the original carriage strain and subsequent carriage strains. The mathematical models predict that if colonization with vaccine types generates long-lasting immune responses to noncapsular antigens, and if these immune responses inhibit acquisition of nonvaccine serotypes even after the host has cleared carriage of the vaccine type [10], then the potential for serotype replacement is greater than if only direct competition occurs. Thus, an important extension of these experiments is to investigate the role of speci®c immunity (antibodies) to capsular and noncapsular antigens in the interactions between serotypes. An additional question is whether carriage of a resident strain in the nasopharynx aects an individual's susceptibility to invasive disease following respiratory exposure to a second strain. Finally, because competition assays are generally the most sensitive way to measure small dierences in ®tness [15], this system provides a way to measure quantitatively the eect of antibiotic resistance mutations and genes (acquired via transformation) on dierent aspects of bacterial ®tness in vivo and the evolution of this eect in strains that are repeatedly passaged in vivo. These experiments considered pairwise interactions between genetically de®ned pneumococcal strains. Simultaneous carriage of two S. pneumoniae serotypes has been documented in several studies, particularly in high-prevalence areas [6,16±18]. Carriage of more than two serotypes has been documented in rare instances, and only in older studies which used the sensitive technique of mouse inoculation to purify minority serotype populations [17,18]. Although it appears to be rare for one individual to carry more than two serotypes simultaneously, there is a highly diverse population of S.
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pneumoniae circulating in any given community. Mathematical models suggest that interactions at the population level among more than two pneumococcal types can be important in structuring the population [8,10], and further modeling work is required to investigate the nature of these population-level interactions. The success of pneumococcal vaccines in reducing disease will depend in part on the way in which pneumococcal populations change under the selective pressures exerted by mass vaccination. Mathematical models, informed by experimental and epidemiological data about the mechanisms of interaction of pneumococci with each other and their hosts, will be an important tool in predicting these changes and their potential eects, in designing clinical trials to detect changes in the pneumococcal population, and in suggesting improvements in vaccine design. Acknowledgements The authors thank R. Malley for valuable discussions and helpful comments on a draft of the manuscript. References [1] Black S, Shine®eld H, Ray P, Lewis EM, Fireman B, Group KPVS, et al. Ecacy of heptavalent conjugate pneumococcal vaccine (Wyeth Lederle) in 37,000 infants and children: impact on pneumonia, otitis media, and an update on invasive disease Ð Results of the Northern California Kaiser Permanente Ecacy Trial. In: 39th ICAAC. San Francisco, CA, 1999. [2] Dagan R, Melamed R, Muallem M, Piglansky L, Greenberg D, Abramson O, et al. Reduction of nasopharyngeal carriage of pneumococci during the second year of life by a heptavalent conjugate pneumococcal vaccine. J Infect Dis 1996;174:1271±8. [3] Eskola J, Takala AK, Kilpi TM, Lankinen KS, Kayhty H. Clinical evaluation of new pneumococcal vaccines: the Finnish approach. Devl Biol Stand 1998;95:85±92. [4] Eskola J, Kilpi T. Ecacy of a heptavalent pneumococcal conjugate vaccine (PncCRM) against serotype-speci®c, culture-con®rmed pneumococcal acute otitis media (AOM) in infants and children. In: 39th ICAAC. San Francisco, CA, 1999. [5] Mbelle N, Huebner RE, Wasas AD, Kimura A, Chang I,
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Klugman KP. Immunogenicity and impact on nasopharyngeal carriage of a nonavalent pneumococcal conjugate vaccine. J Infect Dis 1999;180:1171±6. Obaro SK, Adegbola RA, Banya WAS, Greenwood BM. Carriage of pneumococci after pneumococcal vaccination. Lancet 1996;348:271±2. Dagan R, Givon N, Yagupsky P, Porat N, Janco J, Chang I, et al. Eect of a 9-valent pneumococcal vaccine conjugated to CRM197 (PncCRM9) on nasopharyngeal (NP) carriage of vaccine type and non-vaccine type S. pneumoniae (Pnc) strains among day-care-center (DCC) attendees. In: 38th ICAAC. San Diego, CA, 1998. Lipsitch M. Bacterial vaccines and serotype replacement: lessons from Haemophilus in¯uenzae and prospects for Streptococcus pneumoniae. Emerg Infect Dis 1999;5:336±45. Anderson RM, May RM. Infectious diseases of humans: dynamics and control. Oxford: Oxford University Press, 1991. Lipsitch M. Vaccination against colonizing bacteria with multiple serotypes. Proc Natl Acad Sci USA 1997;94:6571±6. Sanders CC, Sanders Jr WE, Harrowe DJ. Bacterial interference: eects of oral antibiotics on the normal throat ¯ora and its ability to interfere with group A streptococci. Infect Immun 1976;13:808±12. Wu H-Y, Virolainen A, Mathews B, King J, Russell MW, Briles DE. Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice. Microb Path 1997;23:127±37. Levy I, Ettesse C, Rubin LG. Renicillin-resistant pneumococci (PRSP) exhibit less virulence than penicillin-susceptible pneumococci (PSSP) [poster abstract]. Pediat Res 1997;41:125A. Rubin L, Rizvi A. Comparative colonization and virulence of genetically related penicillin-susceptible and penicillin-resistant pneumococci. In: Pneumococcal vaccines for the world conference. Washington, DC, 1998. Lenski RE. The cost of antibiotic resistance Ð from the perspective of a bacterium. In: Chadwick DJ, Goode J, editors. Antibiotic resistance: origins, evolution, selection and spread. Chichester: Wiley, 1997. p. 131±51. Gratten M, Montgomery J, Gerega G, Gratten H, Siwi H, Poli A, et al. Multiple colonization of the upper respiratory tract of Papua New Guinea children with Haemophilus in¯uenzae and Streptococcus pneumoniae. SE Asian J Trop Med Publ Hlth 1989;20:501±9. Gundel M, Okura G. Untersuchungen uber das gleichzeitige Vorkommen mehrerer Pneumokokkentypen bei Gesunden und ihre Bedeutung fur die Epidemiologie. Z Hyg Infekt 1933;114:678±704. Hodges RG, MacLeod CM, Bernhard WG. Epidemic pneumococcal pneumonia. III. Carrier studies. Am J Hyg 1946;44:207± 30.
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Competition among Streptococcus pneumoniae for intranasal colonization in a mouse model M. Lipsitch a, b,*, J.K. Dykes a, S.E. Johnson a, E.W. Ades a, J. King c, D.E. Briles c, G.M. Carlone a a
Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA b Department of Biology, Emory University, Atlanta, GA 30322, USA c Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA Received 29 November 1999; accepted 24 January 2000
Abstract Widespread use of conjugate vaccines against Streptococcus pneumoniae, by reducing carriage of S. pneumoniae serotypes included in the vaccine, may result in an increase in nasopharyngeal carriage of Ð and disease from Ð nonvaccine serotypes of the same species. Mathematical models predict that the extent of such replacement will depend positively on the degree to which carriage of vaccine-type S. pneumoniae inhibits acquisition of nonvaccine-type pneumococci, and may depend negatively on the inhibition of vaccine-type pneumococci by nonvaccine-type pneumococci. We used a mouse model of intranasal carriage of pneumococci to test whether such inhibition occurs between dierent pneumococcal strains. Mice carrying a streptomycinresistant derivative of S. pneumoniae BG9163 (serotype 6B) as a resident strain showed reduced levels of colonization when challenged intranasally by optochin-resistant derivatives of the same strain and of a serotype 23F pneumococcus, BG8826. Inhibition could be overcome by increasing the dose of the challenge strain. Carriage of optochin-resistant BG9163 did not inhibit acquisition of the streptomycin-resistant variant. Colonization by a challenge strain did not signi®cantly aect the level of colonization with the resident strain. These results provide evidence that is consistent with several hitherto untested assumptions of mathematical models of serotype replacement and suggest that a biological mechanism exists that could account for serotype replacement that is observed in clinical trials. The ®ndings provide a basis for further studies of in vivo interactions between strains of S. pneumoniae. Published by Elsevier Science Ltd. Keywords: Streptococcus pneumoniae; Serotype replacement; Nasopharyngeal carriage; Mathematical models; In vivo models
1. Introduction and background Conjugate vaccines against Streptococcus pneumoniae are currently in clinical trials [1±5]. These vaccines immunize against 7, 9, of the 90 known pneumococcal serotypes. Preliminary results from these ongoing trials in infants indicate that these vaccines are highly pro* Corresponding author: Department of Epidemiology, Harvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA. Tel.: +001-617-432-4559; fax: +001-617-566-7805. E-mail address: [email protected] (M. Lipsitch). 0264-410X/00/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 0 4 6 - 3
tective against invasive disease [1] and oer signi®cant protection against pneumonia [1] and otitis media [1,4], from the serotypes included in the vaccine. In addition, the vaccine oers partial protection against nasopharyngeal carriage of the included serotypes [5± 7]. Because nasopharyngeal carriers of pneumococci are the major source for transmission of these bacteria, conjugate vaccines are expected to reduce transmission and prevalence of vaccine-type pneumococci. This reduction in pneumococcal transmission should help to reduce the incidence of disease from vaccine-type pneumococci among unvaccinated individuals in communities where vaccination is used, via herd immunity
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(indirect eects), a hypothesis that is currently being tested in community-randomized clinical trials (K. O'Brien, personal communication). Vaccine-induced reductions in the prevalence of vaccine-type pneumococci may also have the unintended eect of increasing the prevalence of those pneumococcal serotypes not included in the vaccine. This phenomenon of ``serotype replacement'' would be expected to occur if carriage of vaccine-type pneumococci reduces a host's probability or intensity of colonization by nonvaccine-type pneumococci, via competition between bacteria types to colonize the nasopharynx. Preliminary results from clinical trials have suggested that serotype replacement occurs at least at the level of nasopharyngeal carriage [5±7]. Thus far, no increase in disease from nonvaccine-type pneumococci has been observed in the one trial where it has been measured [1]; however, these optimistic results should be tempered by the expectation that serotype replacement may be more pronounced following widespread use of a vaccine in a community than in a clinical trial [8]. Mathematical models are valuable tools for assessing the population-wide consequences of public health interventions such as the introduction of vaccines [9], and in particular for assessing the potential for serotype replacement following vaccination against the pneumococcus [8,10]. Such models may be used to predict, for instance, which biological parameters determine the extent of serotype replacement following vaccination, and how such replacement can best be measured during clinical trials [8,10]. For example, a simple mathematical model of the interaction between
Fig. 1. Predictions of a mathematical model [10] for the maximum possible serotype replacement expected following mass vaccination with a conjugate vaccine. Maximum replacement is de®ned as the absolute increase in prevalence of nonvaccine-type pneumococcal carriage that would be expected if vaccine-type carriage were eradicated. rN (shown on the x-axis) measures the inhibition of nonvaccine-type pneumococcal colonization by vaccine-type carriage; rV measures the inhibition of vaccine-type by nonvaccine-type. The potential for replacement is greatest when nonvaccine-type colonization is strongly reduced by vaccine-type carriage (high rN ) and when vaccine-type colonization is not strongly reduced by nonvaccine-type carriage (low rV ). Parameters for this ®gure: R01=1.8; R02=2.2; c1=1ÿrV; c2=1ÿrN.
vaccine-type and nonvaccine-type pneumococci [10] predicts that the extent of serotype replacement anticipated in a population with high vaccine coverage depends strongly on the ability of vaccine-type pneumococci, carried in the nasopharynx, to reduce the incidence of colonization with nonvaccine-type pneumococci. It also depends, albeit less strongly, on the opposite interaction: the extent to which carriage of nonvaccine-type pneumococci reduces the incidence of colonization with vaccine-type pneumococci. This relationship, as predicted by the mathematical model described in [10], is shown in Fig. 1. The maximum extent of serotype replacement, measured as the absolute increase in the prevalence of carriage of nonvaccine-type pneumococci in a population where vaccination has eliminated carriage of vaccine-type pneumococci, is shown on the vertical axis. The variable on the horizontal axis, rN, measures the strength of competition between a resident, vaccine-type strain and a potentially colonizing nonvaccine-type strain. Speci®cally, rN is de®ned as the reduction in incidence of new colonization by nonvaccine-type S. pneumoniae in individuals currently carrying vaccine-type S. pneumoniae. The dierent curves refer to dierent levels of competition in the opposite direction: rV is de®ned as the reduction in incidence of new colonization by vaccine-type S. pneumoniae in individuals currently carrying nonvaccine-type S. pneumoniae. As Fig. 1 shows, the potential for serotype replacement is greatest if carriage of vaccine-type strongly inhibits acquisition of nonvaccine-type, and is somewhat reduced if carriage of nonvaccine-type strongly inhibits acquisition of vaccine-type. The parameters used in Fig. 1 are arbitrary, but the relationship shown occurs for a broad range of parameter choices. The detailed predictions of the mathematical model have been described previously [8,10]. Several of the model's assumptions, although consistent with present knowledge of the biology and epidemiology of the pneumococcus, have not been formally tested. For example, although there are plausible mechanisms by which dierent strains of pneumococci might compete for colonization of the nasopharynx [11], there are no direct measurements of this phenomenon. As a result, it is not known whether carriage of one pneumococcal strain reduces the probability of acquiring a second strain, or whether it (instead or in addition) reduces the population size attained by a second strain that does successfully colonize a host. To take another example, the model assumes that the population of one strain, once established in the nasopharynx, is not aected by acquisition of a second strain by the same host. In this report, we describe an experimental system in which these assumptions of the model can be tested in vivo. Results are presented to show that, in a mouse
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model, intranasal carriage of one pneumococcal strain can inhibit acquisition of a second pneumococcal strain; that the existence and strength of this inhibition, while repeatable under the same experimental conditions, depends on the strains chosen and the doses administered; and that the inhibition can take the form of reduced probability of colonization and/or reduced bacterial counts in animals that are colonized. Furthermore, in these experiments acquisition of a second strain does not aect the levels of colonization by a resident strain. Thus, we demonstrate that dierent strains of pneumococci compete for intranasal colonization in mice, a mechanism that could result in serotype replacement following widespread use of conjugate vaccines. We provide an experimental system in which these interactions can be assessed systematically for a larger number of strains. 2. Methods 2.1. Bacterial strains S. pneumoniae strains were antibiotic-resistant mutants isolated from strains BG9163 (serotype 6B) and BG8826 (23F) [12]. Optochin-resistant (Opt1) mutants were isolated on tryptic soy agar + 5% sheep's blood plates (SBA) containing 5 mg/l optochin, and streptomycin-resistant (Str1) mutants were isolated on SBA plates containing 200 mg/l streptomycin. Isolates were restreaked on antibiotic plates and then grown to late log phase and plated on both antibioticcontaining and antibiotic-free plates to con®rm the stability of the resistance markers. For use in inoculations, bacteria were grown to optical densities of approx. 0.4 (492 nm) in Todd±Hewitt Broth supplemented with 0.5% yeast extract (THY), and frozen at ÿ708C in 1 ml aliquots in THY+10% glycerol. At the time of inoculation, aliquots were thawed, centrifuged and resuspended in physiologic saline (Gibco) twice, and diluted to the desired density, which was con®rmed by plating. 2.2. Mouse inoculation and sampling Female C57BL/6J (Jackson Laboratories, Bar Harbor, ME, USA) mice aged 5±7 weeks at the beginning of the experiment were used in all experiments. Committee approval was obtained at the Centers for Disease Control and Prevention for animal care and use. Mice were inoculated with de®ned numbers of colonyforming-units (cfu; speci®c numbers given in the Results section) intranasally by pipetting 10 ml of saline containing the bacteria onto the nares [12]. For sampling, mice were anesthetized by an intraperitoneal injection of 0.02 ml ketamine and 0.01 ml xylazine
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diluted in 0.9% NaCl, and sacri®ced by CO2 inhalation. Nasal washes were obtained by washing approx. 0.25 ml of saline through the trachea and collecting the ®rst 0.1 ml of nasal wash as it exited the nares, as described [12]. The wash was collected in sterile tubes already containing 0.1 ml of saline, and stored on ice until diluted and plated on selective media (SBA plates supplemented with 2.5 mg/l gentamicin to suppress growth of organisms other than S. pneumoniae, plus either streptomycin or optochin as above). On occasion (at most one mouse per group) we failed to obtain any nasal wash; this accounts for variations in numbers between groups in the same experiment. 2.3. Experimental protocol For basic competition experiments, groups of 10±12 mice were inoculated intranasally on day 0 with a resident strain or mock-inoculated with saline. On day 3, both groups were challenged with a challenge strain carrying a dierent antibiotic resistance marker. On day 7, mice were sacri®ced and nasal washes obtained and plated on antibiotic selective plates to dierentiate the challenge strain from the resident strain. Competition was de®ned as a reduction in the number of mice colonized with the challenge strain in animals carrying the resident strain vs. controls. Numbers of cfu recovered were compared and statistical signi®cance tested with the two-tailed Mann±Whitney U-test (all mice in which no challenge strain was recovered were considered ties for the lowest rank). Dierences in the number of mice colonized (disregarding level of colonization) were assessed using Fisher's exact test. As noted individually below, the competition experiment was also performed over a 14-day period, with inoculations on days 0 and 7 (rather than 0 and 3) and sacri®ce on day 14. To assess the eect of a challenge strain on the resident strain, the protocol was reversed. On day 0, all mice in two groups were given a resident strain intranasally. On day 7, one group was given saline, the other a challenge strain. On day 14, the mice were sacri®ced, and nasal washes obtained and plated. Competition was de®ned as a reduction in the carriage of resident strain by mice given the challenge strain vs. controls given saline. 3. Results 3.1. Carriage of a resident strain could inhibit colonization by a challenge strain Fig. 2 shows the results of 7-day [Fig. 2(a)] and 14day [Fig. 2(b)] experiments in which BG9163Str1 was the resident strain and the otherwise isogenic
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BG9163Opt1 was the challenge strain. Inocula were 7 104 cfu (resident strain) and 4 104 cfu (challenge strain) for the 7-day experiment, and 6 104 cfu (resident strain) and 4 104 cfu (challenge strain) for the 14-day experiment. Competition was observed both in the 7 and 14-day experiment. In the 7-day experiment, carriage of the resident strain BG9163Str1 reduced the level of carriage of the challenge strain BG9163Opt1 by about 25-fold (median cfu recovered 102.9 vs. 104.3, p < 0.005), with 10/10 mice colonized in each group. In the 14-day experiment, the eect was larger. Challenge strain was recovered from 5/10 mice carrying the resident strain, with a median of 101.4 cfu in the mice carrying any recoverable challenge strain; by contrast, 9/10 control mice were colonized with a median recovery of 104.0 cfu/nasal wash in the colonized mice. The dierence in levels of colonization (with 0 colonization assessed as ties) was highly signi®cant, p < 0.0005 (Mann±Whitney U-test). The dierence in the proportion of mice colonized did not reach statistical signi®cance ( p = 0.15, Fisher's exact test). Similar results were obtained when the challenge strain was BG8826Opt1 (see Fig. 3 below) at approximately the same dose. 3.2. Inhibition was not reciprocal The 14-day experiment described above was repeated with the strains in opposite roles: BG9163Opt1 was the resident strain and BG9163Str1 was the challenge strain. No signi®cant dierence in carriage of the challenge strain between carriers of the resident strain (8/9 mice colonized, median of colonized mice = 104.0 cfu, range 103.6±104.6 cfu) and con-
trols (9/9 mice colonized median = 104.3 cfu, range 103.6±104.6 cfu) was detected. 3.3. The inhibitory eect of a resident strain on acquisition of a challenge strain depended on the dose of the challenge strain Fig. 3 shows the results of 7-day experiments in which BG9163Str1 (inoculum: 8 105 cfu) was the resident strain and BG8826Opt1 was the challenge strain. Note that in contrast to Fig. 2, this experiment used unrelated strains of dierent serotypes, and several doses of the challenge strain were administered, with inocula of 5 104, 5 105 and 5 106 cfu, respectively. Carriage of the resident strain signi®cantly inhibited acquisition of the challenge strain at the lowest challenge dose. At this dose, 7/12 mice carrying the resident strain acquired the challenge strain, with a median of 102.1 cfu among those colonized; 10/11 control mice acquired the challenge strain, with a median of 102.7 cfu among those colonized, p < 0.01 (Mann± Whitney U-test). However, the inhibition did not appear at higher doses of the challenge strain. 3.4. Inoculation of a challenge strain had little or no eect on carriage of a resident strain Fig. 4 shows the results of reciprocal 14-day experiments in which mice were given a resident strain (either BG9163Opt1 or the otherwise isogenic BG9163Str1) at day 0, challenged with the opposite strain or mock-challenged with saline at day 7, and sacri®ced and sampled at day 14. As the ®gures show, there was a small (two to four fold) reduction in the median level of the resident strain in mice that had
Fig. 2. Inhibition of colonization by a challenge strain in mice already carrying a resident strain. Carriage of the resident strain, BG9163Str1 inhibited acquisition of the challenge strain BG9163Opt1 in 7-day (a) and 14-day (b) experiments. Shown are bacterial counts of the challenge strain (®gures give medians of colonized animals) in controls and carriers of the resident strain. N = 10 mice per group. Italic ®gures at the bottom (``NONE'') designate mice from which no challenge strain pneumococci were recovered. P values refer to Mann±Whitney U-test comparing counts of the challenge strain in controls to counts in carriers.
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Fig. 3. Relationship between eect of resident strain (BG9163Str1) on acquisition of a challenge strain (BG8826Opt1) and the dose of the challenge strain. Acquisition of the challenge strain was inhibited in mice carrying the resident strain when the dose of the challenge strain was low, but at higher doses the challenge strain was able to colonize resident strain-carrying mice with equal success to that achieved in controls. N = 12 mice per group except for carriers in 5 104 and 5 106 groups, for which N = 11. Italic ®gures at the bottom (``NONE'') designate mice from which no challenge strain pneumococci were recovered.
been challenged with the challenge strain, but the variation within groups was substantial, and the reduction was not statistically signi®cant. 4. Discussion The experiments reported here demonstrate that S. pneumoniae carried intranasally can interfere with nasal colonization by a second strain of S. pneumoniae to which the animal is exposed. The removal of this interference by reductions in carriage of vaccine-type S. pneumoniae provides a biological mechanism that could lead to serotype replacement following the introduction of a conjugate pneumococcal vaccine into a population. As described in Section 1, mathematical models pre-
dict that the extent of the increase in carriage of nonvaccine-type pneumococci expected following introduction of a vaccine depends on many factors, including the strength of the competitive interaction between pneumococci. These experiments on a few strains demonstrate that several of the basic assumptions of existing mathematical models are correct: (a) carriage of one strain can reduce the level of colonization by a second strain; (b) once established in the nasopharynx, carriage of a resident strain is not aected signi®cantly by acquisition of a second strain; (c) dierent pneumococci vary in their competitive abilities. Another observation that emerges from this study is that antibiotic resistance markers appear to in¯uence components of the ®tness of pneumococci in vivo, where ®tness is de®ned as the ability of a de®ned
Fig. 4. Recovery of the resident strain in control mice vs. mice colonized with a challenge strain, in experiments lasting 14 days. Both strains were antibiotic resistant mutants of BG9163: (a) resident strain: Str1, challenge strain Opt1; (b) resident strain: Opt1, challenge strain Str1. A slight dierence was observed in each case, but neither was statistically signi®cant. N = 10 mice per group. Inocula: 6 104 cfu of Str1 resident, 4 104 cfu of Opt1 challenge; 3 104 cfu of Opt1 resident, 7 104 cfu of Str1 challenge. N = 10 mice per group.
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inoculum to colonize a mouse and grow to high population sizes. This is most obvious from the experiments with otherwise isogenic isolates of BG9163 described in Sections 3.1 and 3.2. In these experiments, we observed that the streptomycin-resistant variant was able to inhibit the acquisition of the optochin-resistant, streptomycin-sensitive variant, but the opposite did not occur. This observation is consistent with previous observations in pneumococci [13,14] and other bacterial species [15], in which antibiotic resistance has resulted in a reduction in bacterial growth rate, virulence and/or competitive ability in vitro and in vivo. One consequence of this observation is that competition studies of S. pneumoniae must be designed in such a way that interpretation of the data does not depend on the assumption that antibiotic resistance markers have no eect on bacterial ®tness. In the present study, this is accomplished by measuring competition as a dierence in the performance of one strain (with the same antibiotic resistance marker) under dierent conditions, for example, comparing colonization with BG9163Opt1 in the presence or absence of BG9163Str1. The experiments described here demonstrate that interference between pneumococci can occur in vivo. These preliminary studies nonetheless have several limitations, most of which can be addressed by further experimental work with a wider range of strains. First, the numbers of mice involved was relatively small, and would not have had the power to detect subtle eects of challenge strains on resident strains. However, the remarkable concordance between the levels of resident strain in mice that were, and were not, challenged with a second strain suggests that such eects, if they occurred, were not large. Second, the small numbers prevented us from determining whether carriage of a resident strain signi®cantly reduced an animal's probability of acquiring a challenge strain, although we were able to show that it reduced the level of colonization (which sometimes took the form of preventing colonization altogether). Third, and perhaps most importantly, the experiments were limited to three strains, which were derived by isolation of antibiotic resistant mutants of two human isolates which were both of serotypes included in the vaccine. Ideally, one would like to use a wider variety of primary human isolates without introduced antibiotic resistance mutations; these studies are currently underway. To use the mathematical model to make more speci®c predictions about the potential for serotype replacement, it will be necessary to measure competitive interactions in a wider range of strains, encompassing a number of vaccine-serotype and nonvaccineserotype pneumococci. For example, if one found that vaccine-type pneumococci were generally better competitors for nasopharyngeal colonization than nonvac-
cine-type pneumococci (meaning that vaccine-type strongly inhibit acquisition of nonvaccine-type but are only weakly inhibited by nonvaccine-type), then the potential for serotype replacement would be relatively high, as suggested by Fig. 1. The experimental techniques described here provide a straightforward way to assess these parameters for any pair of strains, subject of course to the caveats attending any animal model. In addition to investigating the range of interactions among various natural pneumococcal isolates within an individual, the experimental system described here provides the opportunity to address a number of other questions. One such question is the nature of the mechanism(s) of inhibition between strains. This question is not only of biological interest, but also has important consequences for serotype replacement. We have shown that pneumococci compete directly within a host, and the mathematical models indicate that this can lead to serotype replacement when transmission of vaccine serotypes is reduced. If carriage has existed for several weeks, or if the host is already primed to the bacterial antigens, it is possible that speci®c antibodies could be elicited which could have an eect on the original carriage strain and subsequent carriage strains. The mathematical models predict that if colonization with vaccine types generates long-lasting immune responses to noncapsular antigens, and if these immune responses inhibit acquisition of nonvaccine serotypes even after the host has cleared carriage of the vaccine type [10], then the potential for serotype replacement is greater than if only direct competition occurs. Thus, an important extension of these experiments is to investigate the role of speci®c immunity (antibodies) to capsular and noncapsular antigens in the interactions between serotypes. An additional question is whether carriage of a resident strain in the nasopharynx aects an individual's susceptibility to invasive disease following respiratory exposure to a second strain. Finally, because competition assays are generally the most sensitive way to measure small dierences in ®tness [15], this system provides a way to measure quantitatively the eect of antibiotic resistance mutations and genes (acquired via transformation) on dierent aspects of bacterial ®tness in vivo and the evolution of this eect in strains that are repeatedly passaged in vivo. These experiments considered pairwise interactions between genetically de®ned pneumococcal strains. Simultaneous carriage of two S. pneumoniae serotypes has been documented in several studies, particularly in high-prevalence areas [6,16±18]. Carriage of more than two serotypes has been documented in rare instances, and only in older studies which used the sensitive technique of mouse inoculation to purify minority serotype populations [17,18]. Although it appears to be rare for one individual to carry more than two serotypes simultaneously, there is a highly diverse population of S.
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pneumoniae circulating in any given community. Mathematical models suggest that interactions at the population level among more than two pneumococcal types can be important in structuring the population [8,10], and further modeling work is required to investigate the nature of these population-level interactions. The success of pneumococcal vaccines in reducing disease will depend in part on the way in which pneumococcal populations change under the selective pressures exerted by mass vaccination. Mathematical models, informed by experimental and epidemiological data about the mechanisms of interaction of pneumococci with each other and their hosts, will be an important tool in predicting these changes and their potential eects, in designing clinical trials to detect changes in the pneumococcal population, and in suggesting improvements in vaccine design. Acknowledgements The authors thank R. Malley for valuable discussions and helpful comments on a draft of the manuscript. References [1] Black S, Shine®eld H, Ray P, Lewis EM, Fireman B, Group KPVS, et al. Ecacy of heptavalent conjugate pneumococcal vaccine (Wyeth Lederle) in 37,000 infants and children: impact on pneumonia, otitis media, and an update on invasive disease Ð Results of the Northern California Kaiser Permanente Ecacy Trial. In: 39th ICAAC. San Francisco, CA, 1999. [2] Dagan R, Melamed R, Muallem M, Piglansky L, Greenberg D, Abramson O, et al. Reduction of nasopharyngeal carriage of pneumococci during the second year of life by a heptavalent conjugate pneumococcal vaccine. J Infect Dis 1996;174:1271±8. [3] Eskola J, Takala AK, Kilpi TM, Lankinen KS, Kayhty H. Clinical evaluation of new pneumococcal vaccines: the Finnish approach. Devl Biol Stand 1998;95:85±92. [4] Eskola J, Kilpi T. Ecacy of a heptavalent pneumococcal conjugate vaccine (PncCRM) against serotype-speci®c, culture-con®rmed pneumococcal acute otitis media (AOM) in infants and children. In: 39th ICAAC. San Francisco, CA, 1999. [5] Mbelle N, Huebner RE, Wasas AD, Kimura A, Chang I,
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