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Incremental Increase in Fitness Cost with Increased β-Lactam Resistance in Pneumococci Evaluated by Competition in an Infant Rat Nasal Colonization Model

  1. Krzysztof Trzciński1,
  2. Claudette M. Thompson1,
  3. Andrea M. Gilbey2,a,
  4. Christopher G. Dowson2 and
  5. Marc Lipsitch1
  1. 1Departments of Epidemiology and Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts;
  2. 2Infectious Disease Research Group, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom
  1. Reprints or correspondence: Krzysztof Trzciński, Depts. of Epidemiology and Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave., Bldg. 1, Rm. 903, Boston, MA 02115 (ktrzcins{at}hsph.harvard.edu)
 
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Abstract

BackgroundWe evaluated the impact of resistant penicillin-binding protein (PBP) allele acquisition on the ability of penicillin-resistant (PEN-R) pneumococcal strains to compete with penicillin-susceptible (PEN-S) ancestors for upper-respiratory-tract (URT) colonization

MethodsPEN-S serotype 2, 6B, and 9V strains were transformed into derivatives expressing an increasing number of PEN-R PBP forms (2X, 2X-1A, and 2X-1A-2B for serotype 2 and 2X, 2X-2B, and 2X-2B-1A for 6B and 9V). Infant rats were inoculated intranasally with a mix of a PEN-R and PEN-S strains. For consecutive days, samples were collected for assessment of the ratio of PEN-S to PEN-R cells colonizing the URT. The selective index (SI), defined as the change in the natural logarithm of the ratio of PEN-S to PEN-R strains from the inoculum to the nasal-wash samples, quantified differences in fitness

ResultsSIs significantly >0 (indicating a cost of resistant allele acquisition) were observed 4–5 days after colonization in all but serotype 6B pbp2x transfomants. Additional replacements with low-affinity forms of pbp2b and pbp1a genes reduced further ability to compete in all strains

ConclusionsThe cost of penicillin-resistance acquisition for the Streptococcus pneumoniae strain competing with its susceptible ancestor to colonize the URT increases with the number of resistant pbp alleles acquired

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and otitis media. Penicillin resistance emerged among pneumococci >25 years ago [1], and it has become widespread and frequently associated with multidrug resistance [2–5]. At least 20 of 26 epidemic multidrug-resistant (MDR) clones of S. pneumoniae are penicillin nonsusceptible (available at: http://www.sph.emory.edu/PMEN/index.html). Lack of susceptibility to β-lactams in streptococci is associated with changes in penicillin-binding proteins (PBPs) [6]. Although resistant (low β-lactam affinity) forms are known for all 6 PBPs in pneumococci [7, 8], modifications in PBP 1A, 2B, and 2X are required for resistance to penicillin to develop [9]. This resistance has arisen by multiple, independent recombinational events, which indicates the importance of horizontal routes for the emergence and redistribution of low-affinity PBPs [10, 11]

Given that upper-respiratory-tract (URT) carriage is the major event that leads to the transmission of pneumococci, we sought to measure the impact of acquiring low-affinity forms of PBPs on the ability of S. pneumoniae to colonize. The rate at which resistant strains spread in the population is thought to depend on a balance between 2 factors: the volume of drug use in the population and the magnitude of the “fitness cost” of drug resistance in the absence of treatment [12]. If the use of antibiotics is sufficient to offset the fitness cost, then resistant strains will displace susceptible ones, and the rate at which they do so will be greatest if drug use is high and fitness cost is low. If a fitness cost exists, and if antibiotic use decreases to below a level at which it outweighs the fitness cost, then susceptible strains are expected to displace resistant ones. This relationship is complicated by the existence of MDR strains and the consequent ability of one drug class (e.g., macrolides) to select for strains resistant to other classes (e.g., β-lactams); nonetheless, the balance between antibiotic selection and fitness cost is expected to determine the fate of resistant strains. Hence, determining the magnitude of such fitness costs can help us to understand whether reduced antibiotic use is likely to lead to rapid declines in resistance [13]

Most antibiotic resistance in S. pneumoniae (and many other bacteria) is determined by the acquisition of novel alleles (such as PBPs) or genes (e.g., the macrolide resistance determinants ermB and mefA). Because these genetic acquisition events are rare, the major selective effect of antimicrobial use in individuals is not to select newly “created” resistant strains but, rather, to shift the balance between existing resistant and susceptible strains within the individual or the host population. Any use of antibiotics that cures individuals of colonization with susceptible strains leaves resistant strains (in that individual or other hosts) more likely to spread within the host population, given that strains compete to colonize hosts [14, 15]. Thus, one would ideally like to measure the effects of drug-resistance alleles on the ability of strains to colonize and transmit from host to host, because this is a key component of fitness. Given the difficulties of doing this directly in humans or even in animals, one of the most sensitive ways to measure differences in colonizing ability is to cocolonize a host with a pair of strains—resistant and susceptible—and monitor their relative frequency in the host [16]. This is the approach we have taken. We used an infant rat URT in vivo colonization model and laboratory-constructed strains with stepwise changes in β-lactam resistance profiles to measure the impact of the acquisition of resistance genes on the ability of S. pneumoniae to compete for this ecological niche

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Materials and Methods

S. pneumoniae strains, growth conditions, and susceptibility testingThe strains used are described in table 1. Strains 607, 608, 901, and 902 were collected by US Centers for Disease Control and Prevention Active Bacterial Core Surveillance and were donated by Richard Facklam, Chris van Beneden, and Cynthia Whitney [17]. All S. pneumoniae isolates were grown on blood agar base 2 medium (BA medium; Becton Dickinson), supplemented with 5% defibrinated sheep blood (Colorado Serum) at 37°C in 5% CO2, or in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY; Becton Dickinson), and stored frozen at −70°C with 10% glycerol. BA supplemented with appropriate antibiotics was used as the selective medium. MICs of penicillin, cefotaxime, and piperacillin were evaluated by E-test (AB Biodisk) [23]. Multilocus sequence typing (MLST) was performed as described elsewhere [24]

Construction of resistant variants of S. pneumoniae.  Serotype 6B strain 608 and 9V strain 902, both of which are resistant to penicillin (PEN-R), were used as donors of pbp gene fragments to construct β-lactam–nonsusceptible variants of the same serotype strains, 607 and 901. Penicillin-susceptible (PEN-S) strains were sequentially transformed with polymerase chain reaction (PCR) products of resistant alleles of pbp2x, pbp2b and pbp1a amplified with primers described by Gherardi et al. [25], and resistant variants were selected on BA with 0.5 mg/L cefotaxime and 0.25 mg/L piperacillin for both serotype 6B and 9V isolates and 0.4 and 0.3 mg/L penicillin for serotype 6B and 9V variants, respectively. After each transformation step, up to 20 randomly picked resistant variants were evaluated by agar dilution for growth at concentrations of β-lactam that were 2, 3, and 4 times higher than what was used for transformant selection [26]. The PEN-S serotype 2 strain D39 was transformed into PEN-R variants with plasmid-cloned low-affinity forms of pbp2x, pbp1a and pbp2b of serotype 19A strain 159 of the Hungary19A-6 epidemic clone, as described elsewhere [19]. Subsequent transformants were selected on BA supplemented with cefotaxime at concentrations of 0.016–2 mg/L (D392x and D392x1a) and with penicillin at concentrations of 0.06–16 mg/L (D392x1a2b) [18]. Transformation was performed as described by Pozzi et al. [27]

Genetic characterization of PBP transformantsFor isolates of serotype 6B and 9V, transformants were screened for the size of recombinational replacements using restriction fragment–length polymorphism analysis of PCR-amplified fragments of the particular pbp from these, recipient, and donor strains, to identify variants with the largest replacement using AluI, DdeI, HaeIII, HinfI, MseI, RsaI, and Tsp509I fingerprints [28]. Fragments of pbp genes amplified from transformants carrying resistant alleles of all 3 genes were sequenced and compared with sequences observed in donors and recipients, to identify sites of recombinational cross-over. The effect of changes in PBPs on the in vitro growth rate was evaluated as described elsewhere [29] and by monitoring the changes of the ratio of PEN-R to PEN-S cells in coculture of 6072x2b1a and 607 strains. For this, 50 mL of THY was inoculated with 1×102 cfu/mL each of PEN-R and PEN-S serotype 6B variants, and the ratio of resistant to susceptible cells was measured every hour, for 11 h, by inoculating BA and BA plates supplemented with 0.05 mg/L cefotaxime

Construction of control strains to assess the effect of transformation on ability to competeTo control for the possibility that fitness differences between more-resistant transformants and parental strains might be caused by transformation effects rather than by the specific genetic changes involved, we first selected a streptomycin-resistant mutant of 607, using BA supplemented with 200 mg/L streptomycin, and transformed it into cefotaxime-resistant variant 607S2x using the protocol described above. We then introduced the Janus cassette [20] into the cbp3 gene of 607S2x by transforming it with a PCR product of CP1296 amplified with the DAM313-DAM316 pair of primers, as described by Sung et al. [20], and selecting for kanamycin resistance and streptomycin susceptibility, to create strain 607S2xJ. We then created strain 607S2xcbp3, which was designed to be isogenic to 607S2x but underwent 2 additional transformations, by transforming 607S2xJ with the chromosomal DNA of 6072x and selecting for resistance to streptomycin and susceptibility to kanamycin and, thus, loss of the Janus cassette

Rat modelAll strains used in the rat model were tested for their ability to colonize 3-day-old, outbred Sprague-Dawley infant rat pups (Jackson Laboratory). To minimize the short-term effects of transformation and allow adaptation to colonization of rats, all strains used in competition experiments were first inoculated intranasally into rats (1×106 cfu) and recovered 4 days later. For this, each rat’s nasopharynx was washed by the installation of 20 μL of saline into the left naris and recovery of fluids expelled from the right naris. These rat-passed strains were frozen in the midlogarithmic phase in THY with 10% glycerol, then centrifuged at 14,000 g for 3 min and resuspended in PBS at the time of competition experiments. For the competition experiments, 3- or 4-day-old infant rats were inoculated intranasally with a total of 1.3–2.2×106 cfu of susceptible cells mixed with resistant cells in a 10:1 ratio. We then measured changes in this ratio in nasal-wash samples collected for up to 5 consecutive days. Each experiment was performed in a litter of infant rats, with pups randomized to dams. Individual pups were marked for tracing through the course of the experiment. Nasal-wash samples were collected almost daily, and 30 μL of undiluted sample and 5-fold dilutions down to 1×5-3 were used to inoculate BA supplemented with 2.5 mg/L gentamicin (BA-gent), to evaluate the total number of S. pneumoniae colony-forming units, and gentamycin plus 0.05 mg/L cefotaxime (BA-gent-ctx), to evaluate the number of β-lactam–nonsusceptible colony-forming units. In the series of in vivo competition experiments, PEN-S recipient strains were competing against their resistant variants and against donors of resistant PBPs alleles

Statistical analysisFor each rat r on day d the density of colonization (colony-forming units/nasal-wash sample) was estimated by serial dilution, both on nonselective (BA-gent, grd) and selective (BA-gent-ctx, brd) plates. The measure of resistant versus susceptible colonization in a given rat on a given day was defined as xrd, and the natural logarithm of the ratio of susceptible to resistant colony-forming units was defined as xrd=ln([grd-brd]/brd). The corresponding log ratio in the inoculum, xI was determined in the same way. The selective index (SI) for rat r, srd was calculated as srd=(xrd-xI). Larger values of srd indicate larger decreases in the representation of the resistant strain. The mean and SE of this SI were calculated, and the null hypothesis that the SI was 0 was tested by a 2-tailed t test

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Results

Genetic characterization of S. pneumoniae strainsCharacteristics of strains used in the study are summarized in table 1. Serotype 6B strain 608 and 9V strain 902 used as donors of resistant pbp alleles were identified by MLST as members of the pandemic Spain6B-2 and Spain9V-3 clones, respectively. PEN-S strains 607 and 901 were of MLST sequence types (STs) identified in PEN-S strains only (available at: http://spneumoniae.mlst.net/)

Analysis of sequences of pbp2x, pbp2b and pbp1a in serotype 6B and 9V transformants confirmed the extended replacements covering the whole transpeptidase (TP) domain in all pbp genes modified except for pbp1a of the 9012x2b1a isolate (figure 1), in which the fragment replaced lacked the first 1 and the last 12 amino acid substitutions within the TP domain. Replacements observed in pbp2b and pbp2x of both serotype 6B and 9V transformants and in PBP1A of the serotype 9V variant probably emerged in single recombinational events, each of which covered a large fragment of the open reading frame (ORF). Nucleotide sequence analysis of the 6B serotype pbp1a revealed that at least 2 replacements took place within the fragment analyzed. As the result, remnants of the susceptible allele were present within the TP domain of the hybrid (missing L583M and A585V substitutions in the pbp1a of 6072x2b1a)

Figure 1
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Figure 1

Graphical presentation of amino acid substitution within penicillin-binding proteins (PBPs) 1A, 2B, and 2X in Streptococcus pneumoniae serotype 6B and 9V strains constructed in the course of the study. Polymerase chain reaction products of fragments of pbp loci corresponding to these used for transformation were sequenced and analyzed. Included are the whole 2160-bp open reading frame (ORF) of the pbp1a gene, the fragment downstream from the 613 nt (codon 205) of the pbp2b ORF of 2043 bp, and the fragment downstream from the 280 nt (codon 94) of the pbp2x ORF of 2253 bp (GenBank accession nos. DQ190413–DQ190418 and DQ227571–DQ227582). The top sequence shows amino acids of penicillin-susceptible isolate TIGR4 (position in the protein as marked above), substitutions of which were identified in isolates analyzed elsewhere [21]. Only differences between TIGR4 and strains analyzed are indicated, and dashes indicate that the amino acid at that particular position was identical with TIGR4 sequence. Strains are presented in the order: susceptible recipient, final transformant (T), and the resistant donor of the pbp gene. Asterisks show the domain’s transpeptidase (TP) active sites (Sx2K, SxN, and KT(S)G, respectively). Symbols “>” and “<” mark first and last replacement within TP domain of particular pbp2x, pbp2b [6], and pbp1a gene [22]. Sequences for transformed variants were determined in isolates passed through the infant rat colonization step before use in an in vivo competition model experiment

In 6072x2b1a, point mutations within pbps were identified in addition to recombinational replacements. This strain had an A→G mutation at nucleotide position 1042 of pbp2x ORF that led to an I348V substitution and 2 mutations within the pbp1a ORF: a T→A mutation at position 1938 that led to an F646L substitution and a silent mutation at position 1989 (A→G). Both amino acid substitutions due to point mutations took place within the TP domain of the enzymes

Phenotypic profiles of transformantsAlterations in PBPs of isolate 607 were sufficient to establish in 6072x2b1a a profile of β-lactam resistance identical to that observed in strain 608, the donor of pbp genes. In the other 2 serotype variants constructed, β-lactam MICs for triple-pbp transformants increased but did not match those of donor strains. As expected, recombinational replacements within pbp2x, pbp2b and pbp1a led to gradual increases in β-lactams MICs in all 3 strain backgrounds (table 1). There was no significant impact of recombinational replacements on the ability of the strains to grow in vitro for both 607 and D39 strain variants. Analysis of growth curve slopes showed no differences between ancestral strains and all variants constructed during the course of the study (P>.4 and P>.17, for serotype 6B and serotype 2 variants, respectively). In vitro growth curves for strain 901 and its derivative proved irreproducible and were not analyzed. In the coculture experiment with 607 and 6072x2b1a strains, the ratio of the resistant to susceptible cells at the early stationary phase and 11 h from the culture start point increased slightly (by 26%), which indicates that there was no detectable in vitro cost of PBP acquisition detected in 6072x2b1a

Figure 2
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Figure 2

Changes in the natural logarithm (LN) of the ratio of resistant variant colony-forming units to susceptible ancestor strain colony-forming units for a set of 3 different Streptococcus pneumoniae isolates and their transformants, as observed in an infant rat intranasal colonization competition model. Graphs represent differences in ratios at day 5 (serotype 6B strain, 607 variants; serotype 9V strain, 901 variants), and day 4 (serotype 2 strain, D39 variants) after inoculation. Labels on the X-axis indicate which genes were modified in the ancestor strain by in vitro recombinational replacement, as described in Material and Methods. The first column shows results for strain 607S2xcbp3 competing against its ancestor 607S strain in the experiment that assessed the effect of neutral transformations on the ability to compete. A detailed description of strains is presented in table 1. The no. of infant rats in every group is depicted at the bottom. Changes in this ratio were evaluated for each individual animal. Solid lines indicate group means; changes in the ratio of resistant to susceptible colony-forming units are marked when statistically significant (*P<.05 and **P<.001, 2-tailed, 1-sample t test of the null hypothesis that the mean logarithm ratio is 0 for a group)

Figure 3
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Figure 3

Results of competition in the infant rat intranasal colonization model, with a modified ratio of resistant to susceptible cells in the inoculum. For isolates 6072x and 9012x competing with β-lactam–susceptible ancestors 607 and 901, respectively, the ratio of resistant to susceptible cells was 1:1; for variants 6022x2b1a and 9012x2b1a competing with susceptible variants, the same ratio was 10:1. Graphs represent changes in the natural logarithm (LN) of the ratio of resistant to susceptible colony-forming units as observed on day 5 after infants’ inoculation. Labels on the X-axis indicate which genes were modified. The no. of infants in each group is depicted at the bottom. Solid lines indicate means; changes in the ratio of resistant to susceptible colony-forming units are marked (*P<.05 and **P<.001, 2-tailed, 1-sample t test of the null hypothesis that the mean logarithm ratio is 0 for a group)

Table 1
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Table 1

Streptococcus pneumoniae strains used in the study

Increasing numbers of pbp substitutions and greater SIs in vivoFigure 2 shows the changes in the ratio of β-lactam resistant to susceptible variants observed 4 or 5 days after inoculation in competition experiments. SIs significantly >0 (indicating a cost of the resistant alleles) were observed for every isolate in competition with its susceptible parent except for strain 6072x (mean ± SE SI, 0.97±0.64; P=.15). In general, additional replacements of PBP genes with low-affinity alleles further reduced the ability of the variant to compete with susceptible strain (figure 2)

After 5 days, a significant decline in the resistant strain was noted for the derivative of 607 carrying low-affinity forms of both pbp2x and pbp2b (mean ± SE SI, 3.35±0.92; P = .0045) and for the variant carrying low-affinity forms of all 3 pbps (mean ± SE SI, 4.75±0.87; P=.0003). A mean ± SE SI of 3.24±0.51 (P<.0001) was observed for the pbp2x transformant of the serotype 9V strain at day 5, whereas its double and triple transformants disappeared from all rats by day 4. For D39 strain variants, results were available only up to day 4 of colonization. Mean ± SE SIs of 1.99±0.62 (P=.0067), 2.98±0.88 (P=.006), and 4.49±0.75 (P<.0001) were obtained for the D392x, D392x1a and D392x1a2b variants, respectively

To rule out the possibility that competitive inferiority of PEN-R cells resulted from their initial low numbers in the inoculum, additional experiments were performed for isolates 6072x and 9012x mixed at a ratio of 1:1 with PEN-S variants, and for 6072x2b1a and 9012x2b1a mixed with PEN-S at a ratio of 10:1 (all in groups of 5–6 rats). Results (figure 3) were consistent with those found at an initial ratio of 1:10; in 1 case (9012x), the resistant strain decreased more when starting at a higher ratio (P=.003, Mann-Whitney U test), but this reflected greater sensitivity of the assay due to the higher starting inoculum of the declining strain

Competitive differences between pbp donors and susceptible strainsFitness differences in our model were also evaluated for the strains used as donors of resistant alleles, compared with the susceptible recipient strains. In a competition between the donor and recipient of type 6B, the resistant donor strain (608) was detected in all rats 1 day after inoculation, but a large and statistically significant SI (mean ± SE, 3.43±0.55; P=.0004) was observed; the next day, strain 608 was undetectable. In vitro growth curves did not differ between strains 607 and 608 (P=.92). Results for strain 902 competing against strain 901 were similar to these observed for serotype 6B strains, with only 7 of 12 rats still colonized by the resistant strain at day 2 (mean ± SE SI, 6.03±0.58; P<.0001) and no detectable strain 902 at day 4. No significant difference in fitness was identified in vivo between strain 159 and the susceptible recipient strain D39 by day 4, with 11 of 14 rats still colonized and a mean ± SE SI of −1.24±1.12 (P=.75). Because of the counting methods used, measurements of increased ratios of resistant to susceptible strains are prone to error. Hence, this increase in the proportion of resistant strains (corresponding to a negative SI) should be interpreted with caution and seen as at least a confirmation that the proportion did not decline

Competitive differences: not artifacts of transformation To control for the possibility that the costs observed was due to an effect associated with transformation per se, we competed strain 607S2xcbp3 against strain 607S in our in vivo model. The decline in the resistant subpopulation was not significant (mean ± SE at day 5, 0.39±0.19; P=.06), a result similar to that reported for 6072x competing with 607 (figure 2)

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Discussion

The present study was designed to evaluate the effect of acquiring β-lactam–resistant alleles of PBP genes on the ability of S. pneumoniae to colonize an animal host. The first step was to create isogenic variants of pneumococcal strains differing only in their resistance to β-lactams. We used donors and recipients of serotypes 6B and 9V because both types are common among both penicillin-susceptible and -resistant clinical and carriage strains in humans. Several steps were taken to minimize the artifactual creation of strains with high fitness costs. Homologous serotype strains were used as donors of PBPs, to avoid the possibility (to our knowledge, only hypothetical) that there might be functional interactions between the capsular polysaccharide and PBPs that could affect a strain’s ability to colonize. Widely disseminated pandemic clones were used as donors of low-affinity forms of PBPs. We assumed that, because pandemic strains have been so successful in their spread, PBP alleles present in both strains 608 and 901 S. pneumoniae would be most likely to carry a relatively low biological cost for these isolates. Variants with the largest replacements within TP domains were selected to minimize the possibility of creating hybrids of diminished physiological fitness. On the basis of the 3 variants constructed for each strain, it appears that, with every consecutive resistant pbp allele introduced, the ability to compete with the susceptible variant further diminished in our model

Reduced fitness, relative to the corresponding susceptible strain, was clearly detected in 2 of 3 resistant donor strains competing in our model. Both strains 607 and 901 outcompeted their respective, fully resistant, serotype-matched donor strains. The opposite was observed for the 159 isolate competing against strain D39; however, strain D39 was the only strain in this set of S. pneumoniae isolates with a long history of being used in laboratory settings, so this difference may reflect declines in the colonizing ability of strain D39 after laboratory passage [30]

Most S. pneumoniae strains resistant to penicillin belong to a limited group of capsular serotypes, which are frequently found in young children [31, 32]. Colonization of the URT is a necessary step before pneumococcal infection in humans. Penicillin resistance was associated with diminished mouse virulence [33] in one study, although the serotype associations of resistance prevented an inference about the causal role of PBP changes in virulence. Virulent isolates transformed into variants resistant to penicillin showed reduced virulence in a murine peritonitis model [34], although a single passage through mice restored the strain to its original virulence

The impact of drug-resistance mutations or gene acquisition on components of bacterial fitness, such as in vitro or in vivo growth rate or competitive ability, has been studied repeatedly [13]. Nearly all such studies have found measurable costs frequently accompanying the acquisition of drug resistance, but, in several cases, compensatory mutations, either intragenic or extragenic, reduced these costs [13]. To the extent that such compensation occurs in nature or that cost-free strains are able to have arisen even in rare cases [35], the ability to reduce drug resistance by reducing antibiotic use may be compromised. Resistance was associated with a fitness cost in 3 different sets of S. pneumoniae strains. The cost increased with the degree of resistance conferred by additional pbp gene substitutions. We found no evidence of compensation for the costs of carrying resistant pbp alleles, but such compensation might arise if the experiments were continued over longer periods. Two multiply resistant donor strains were competitively inferior to their serotype-matched susceptible counterparts when they competed in the absence of drug. If these results could be extrapolated to humans, this would suggest that the success of these 2 resistant strains, which belong to pandemic clones, is due to the widespread use of antibiotics rather than to other intrinsic properties of these strains that make them good colonizers. This interpretation is consistent with evidence that geographic areas of high antibiotic resistance tend to have high levels of resistance in multiple serotypes [4] and species [36], which suggests that antibiotic selection, rather than simply the success of particular resistant clones, accounts for high levels of resistance. It is possible, however, that, in humans, pandemic clones are highly fit and are able to spread widely even in the absence of drugs and that our rat model fails to capture some aspect of this high fitness

The acquisition of low-affinity forms of PBP, in the strains examined, impedes the ability of S. pneumoniae to colonize the URT. This burden grows with the number of PBPs altered and was identified in all strains constructed during the course of the study. This finding supports the hypothesis that selective pressure from antibiotics is necessary to maintain presence of resistant strains in pneumococcal population colonizing and infecting humans

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Acknowledgments

We thank Richard Facklam, Cynthia Whitney, Chris van Beneden, and Donald A. Morrison, for strains

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Footnotes

  • ↵Presented in part: 4th International Symposium on Pneumococci and Pneumococcal Diseases, Helsinki, Finland, 9–13 May 2004 (abstract RES-04)

    Potential conflicts of interest: none reported

    Financial support: National Institutes of Health (grant 1R01AI48935); Ellison Medical Foundation (New Investigator Award to M.L.); British Society for Antimicrobial Chemotherapy (grant to A.G.)

  • ↵Present affiliation: Micropathology Ltd., University of Warwick Science Park, Coventry, United Kingdom

  • Received August 1, 2005.
  • Accepted November 11, 2005.
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  1. J Infect Dis. (2006) 193 (9): 1296-1303. doi: 10.1086/501367
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