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Journal of Clinical Microbiology, January 2007, p. 97-101, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01658-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of In Vitro Biofilm-Associated Pneumococcal Phase Variants of a Clinically Relevant Serotype 3 Clone

M. Catherine McEllistrem,^* Jennifer V. Ransford, and Saleem A. Khan

University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Received 10 August 2006/ Returned for modification 20 September 2006/ Accepted 20 October 2006

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An increasing proportion of children with acute otitis media due to Streptococcus pneumoniae have serotype 3 infections since licensure of the seven-valent pneumococcal conjugate vaccine. These serotype 3 strains are genetically related by molecular subtyping. During otitis media with effusion and recurrent otitis media, biofilms commonly develop. Pneumococcal in vitro biofilms are comprised of phase variants that differ in colony morphology. By using a representative strain of the mucoid serotype 3 clone, rough phase variants with a diverse array of mutations were detected in biofilms formed in vitro. Most phase variants had mutations in the cps3D gene, the first gene of the capsular operon. Eleven had single nucleotide polymorphisms (SNPs) in the cps3D gene, one had an SNP in the –10 promoter, and three had large deletions in the cps3D gene. Reversion to the mucoid phenotype was associated with reversion of the mutation in the cps3D gene. Unlike the phase variants detected in the nasopharynx, which have at least 20% of the parental amount of capsule, the in vitro biofilm-associated phase variants had 12% of the parental amount of capsule, as determined by capsule enzyme-linked immunosorbent assays. Using real-time reverse transcription-PCR, we determined that capsule expression in the phase variants was likely regulated at multiple levels. These in vitro phase variation data, which underscore the plasticity of the pneumococcus, need to be confirmed with in vivo analyses of the middle ear mucosa during otitis media.

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Among cases of acute otitis media (AOM) due to Streptococcus pneumoniae, serotype replacement is occurring since licensure of the seven-valent pneumococcal conjugate vaccine (PCV7) (23, 24). For example, the proportion of cases of AOM due to a serotype 3 clone of the sequence type 180 complex by the multilocus sequencing typing scheme (www.mlst.net) (14, 24) increased from 3% (5/182) in 1999 to 11% (9/82) in 2002 (P < 0.01) (24). Akin to the incidence of AOM, the incidence of serotype 3 invasive disease is also increasing among children with invasive disease, despite an overall decline in the numbers of invasive infections (9).

The capsule polysaccharide is a key virulence factor of S. pneumoniae, and strains with at least 20% of the parental amount of capsule can colonize the nasopharynx (21). Phase variants, which differ in the amount of capsule present, contribute to the pneumococcus's ability to adapt to the environment and evade the host response. The opaque variants have more capsule present than the transparent variants (20). Two subpopulations of phase variants are present in the nasopharynx. On the nasal mucosa surface, transparent phase variants predominate; however, within the nasal mucosa, the majority of the strains are opaque phase variants (7).

Human and animal models clearly demonstrate that otitis media with effusion and recurrent otitis media are biofilm diseases (13, 17). The bacteria, including S. pneumoniae, cannot usually be detected in the biofilms by culture; instead, PCR, fluorescence in situ hybridization, and immunostaining are often used to identify the pathogen (13, 17). Due to the difficulty associated with the culture of bacteria from in vivo biofilms, in vitro biofilm models have been used to study the characteristics of the bacteria in this environment (1, 33). By using an in vitro serotype 3 biofilm model, capsule production was shown to decrease over time (1). While the presence of phase variants was not determined in that study, Waite et al. demonstrated that acapsular variants in an in vitro serotype 3 biofilm increased over time (33).

Acapsular strains of serotype 3 have been found to have mutations in the first gene of the capsular operon, cps3DSUM (12). The capsular operon contains only two type-specific genes: cps3D and cps3S. The cps3D gene encodes a UDP-Glc dehydrogenase that converts UDP-glucose (Glc) into UDP-glucuronic acid (GlcA) (12). This enzyme has an NAD-binding domain spanning residues 2 to 29 and an active site spanning residues 251 to 263 (12). The cps3S gene encodes a processive β-glycosyltransferase that catalyzes the formation of glycosidic linkages to polymerize UDP-Glc and UDP-GlcA, thereby generating the cellobiuronic acid capsule (4, 5). The last two genes in the operon, cps3U and cps3M, have genomic homologues, pgm (26) and galU (18), respectively. Therefore, functional cps3U and cps3M genes are not required for serotype 3 capsule biosynthesis (3, 18, 26). Acapsular variants have been shown to arise due to mutations in the galU (18) and pgm (19) genes.

In this study, we generated biofilms using a clinically relevant serotype 3 clone to assess the genotypic diversity and level of capsule regulation among these phase variants. The phase variants had a diverse array of mutations, and capsule production appeared to be regulated at multiple levels. The marked reduction in capsule production in these phase variants suggests that the biofilms generated in the middle ear during otitis have different subpopulations of pneumococci than the nasopharynx.

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Strain description. A representative of the serotype 3 clone that is associated with a higher proportion of cases of acute otitis media since licensure of PCV7 (24) was used for these studies.

Biofilms. S. pneumoniae strains were grown in Todd-Hewitt broth (THB; Fisher, Pittsburgh, PA) or on 5% sheep blood agar (SBA; Fisher) at 34°C with 5% CO₂. Nine different plate-based biofilms were generated. Each filter (0.2 µm/25 mm; Sterlitech) was seeded with 10⁶ colonies of a representative of the serotype 3 clone and incubated on 5% SBA plates overnight at 34°C. Each biofilm was vigorously "washed" daily by the addition of phosphate-buffered saline (PBS) and scraping of the filter against the agar in an effort to remove adventitiously associated cells (16). The biofilm was placed on a new 5% SBA plate after each wash. After 4 to 7 days, the biofilms were vortexed and the bacteria were grown overnight on 5% SBA. The capsular operon's promoter, cps3D, and cps3S genes were sequenced for a sample of 46 serotype 3 rough phase variants; and the sequences were compared to those of the genes of the mucoid serotype 3 clone.

DNA. DNA was extracted by suspending bacterial colonies in 500 µl of PBS and boiling for 20 min. PCR was performed with an aliquot of the supernatant. The primers are listed in Table 1. A 30-µl reaction mixture containing 1.5 mM MgCl₂, 0.33 µM each primer, 25 µM each deoxyribonucleotide, 0.5 U of the thermostable DNA Taq polymerase mixture, 3 µl of 10x buffer, and 2 µl of DNA template was used. PCR was performed in a 9700 thermal cycler (Perkin-Elmer, Boston, MA). The PCR products were sequenced by using a BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) and were run on a 3730 DNA sequencer (Applied Biosystems). The cps3D gene was amplified with published primers (33) and internal primer sequences (Table 1). For the two rough phase variants without a detectable mutation in the promoter or coding region of the cps3D gene, the cellular phosphoglucomutase (pgm) and the uridine diphosphoglucose (UDP-Glc) pyrophosphorylase (galU) genes were also sequenced (Table 1).

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TABLE 1. Primer sequences

RNA. RNA was isolated from the serotype 3 clone and seven rough phase variants with TRIzol (Invitrogen, Carlsbad, CA) after the cultures were grown to mid-exponential phase at 37°C in THB. Each sample was then purified by using an RNAeasy Mini kit and an RNase-free DNase set (QIAGEN, Valencia, CA). To ensure that contaminating DNA was not present, the final RNA preparation was tested by standard PCR amplification with cps3S-specific primers which annealed to positions 2290 to 2319 and 3075 to 3104 of the sequence with GenBank accession number with U15171. The RNA samples were also run on a denaturing gel to ensure the integrity of the RNA.

Quantitative real-time reverse transcription-PCR. The amounts of cps3DSUM transcript between the selected rough phase variants and the serotype 3 clone were compared by quantitative real-time reverse transcription-PCR (2, 29). Negative control reactions, which consisted of reaction mixtures without reverse transcriptase, were also performed for each strain. The 16S rRNA gene was used as an internal control for data normalization. Primer-probe sets were selected by using Primer Express software (Applied Biosystems). Reverse transcription was performed with 800 ng of RNA in a 100-µl reaction volume by using a high-capacity cDNA archive kit (Applied Biosystems), according to the manufacturer's protocol. To quantify the mRNA, the cDNA templates were diluted 10-fold in 1x PCR buffer and used in subsequent experiments. Quantitative real-time PCR was performed with TaqMan universal master mix (Applied Biosystems) on an ABI Prism 7900HT instrument with the following conditions: 95°C for 12 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The primers were used at concentrations of 250 nM and the probes were used at concentrations of 100 nM in a 25-µl reaction mixture.

The results were calculated by using the comparative critical threshold (C_T) method (User Bulletin No. 2 [http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf]; Applied Biosystems), in which the amount of target is normalized to relative to that of a reference (that of the serotype 3 clone), which consisted of an internal calibrator target RNA (16S rRNA). The cps3S primers amplified positions 2582 to 2686, and the probe targeted the sequence from positions 2613 to 2636. The 16S rRNA primers amplified positions 1186 to 1258 of the genome of TIGR4, and the probe targeted the sequence from positions 1208 to 1229. For each strain, three replicates were performed with duplicate and independent RNA samples. The change (n-fold) in the level of expression of the cps3DSUM transcript was relative to the level of expression of the serotype 3 clone. Statistical analyses were performed by an unpaired Student t test. Significant differences (P < 0.05) in the change in C_T for each rough phase variant compared to that for the serotype 3 clone were determined by using the unpaired t test.

Capsule determination. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) technique was used to determine the quantities of capsular polysaccharide (10) by using 5-ml cultures grown to an optical density at 600 nm of 0.5 in THB and stored at –80°C. One hundred microliters of 1 µg/ml type 3 polysaccharide from ATCC was added to each well of a Costar 96-well plate (Fisher), and the plate was incubated overnight at 4°C. Unless otherwise noted, each additional incubation step was performed in an incubator at room temperature for 2 h. The plate was blocked with 200 µl blocking buffer (1% bovine serum albumin in PBS [154 mM NaCl, 2.22 mM Na₂HPO₄, 1.06 mM KH₂PO₄, pH 7.4]). Except after the blocking step, the plate was washed three times with 0.05% Tween 20 in PBS (pH 7.4) between each step. The bacterial cultures were heat killed for 20 min at 65°C and centrifuged (14,000 x g) for 20 min. The pellets were resuspended in 500 µl blocking buffer. Fifty microliters of serial dilutions of bacteria was first added to the plate, followed by the addition of 50 µl of a 1:20 dilution of serotype 3 mouse immunoglobulin M monoclonal antibody (monoclonal antibody Hyp3M6, provided by the laboratory of Moon Nahm, University of Alabama, Birmingham). After incubation, 100 µl of a 1:3,000 dilution of goat anti-mouse alkaline phosphatase conjugate (Sigma, St. Louis, MO) in blocking buffer was added and the mixture was incubated for 1.5 h. The plates were developed with p-nitrophenyl phosphate (Sigma), and the optical density at 405 nm was read. The concentration that produced 50% inhibition was determined from an interpolated standard curve for the serotype 3 polysaccharide (10). The lower limit of detection of the purified type 3 capsule was 0.05 µg/ml. The experiments included at least three determinations, performed in duplicate.

Reversion frequency. Single colonies of five rough phase variants were grown on 5% SBA overnight in 5% CO₂ at 37°C. One CFU was suspended in 2 ml of THB and incubated for 5 to 6 h in 5% CO₂ at 37°C until mid-exponential phase was reached. Two hundred microliters of a 10^–5 dilution was plated onto 100 5% SBA plates, and the plates were incubated overnight at 37°C. The proportion of strains with a mucoid phenotype per the total number of CFU examined yielded the reversion frequency. Sequencing of the cps3D gene was performed for each strain with a presumed reverted phenotype.

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Genotype and capsule production of serotype 3 rough phase variants. We detected 10¹¹ to 10¹² colonies per biofilm; overall, 24% (247/1,036) of the biofilms visualized had a rough morphology after 4 to 7 days of biofilm maturation. Among 46 rough serotype 3 phase variants characterized from nine different biofilms, 15 genotypically unique rough phase variants were detected. Eleven had single nucleotide polymorphisms (SNPs) in the cps3D-coding region, one had an SNP in the putative –10 promoter, and three had large deletions in cps3D. The deletion in one of the last group of rough phase variants extended into the 5' end of the cps3S gene (Table 2). Among the rough phase variants with point mutations, both transitions and transversions were detected. For two rough phase variants, no mutations were reproducibly detected in the cps3D, cps3S, pgm, or galU gene compared to the sequences of the genes of the serotype 3 clone. Three rough phase variants with missense mutations had the most capsule, generating from 3 to 12% of the parental amount of capsule (Table 2). All rough phase variants had 12% of the amount of capsule present in the serotype 3 clone (180.4 ± 8.6 µg/ml).

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TABLE 2. Genotypes of the 46 rough phase variants compared to those of the mucoid serotype 3 clone

Control of capsule production among seven rough phase variants. To determine whether capsule production was regulated during transcription, the cps3DSUM transcript levels of seven rough phase variants were compared to the transcript level of the serotype 3 clone. The rough phase variants included two strains with missense mutations (I297T and V11I), a strain with a nonsense mutation (Y353stop), a strain with a large deletion (200 to 261 residues), a strain with a –10 promoter down-mutation (CATAAT instead of TATAAT), and two rough phase variants with unknown mutations. The amount of steady-state 16S rRNA levels varied less than twofold among each of the rough phase variants and the serotype 3 clone. The amounts of the steady-state cps3DSUM transcript levels of the serotype 3 rough phase variants with unknown, missense, and nonsense mutations were significantly greater than that of the serotype 3 clone. In contrast, the serotype 3 clone had a >20-fold higher level of expression of the cps3DSUM transcript compared to that of the rough phase variant with the promoter mutation (Fig. 1).

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FIG. 1. Expression of cps3DSUM. Data are expressed as the fold difference for each serotype 3 rough phase variant compared to the value for the serotype 3 clone. The phase variant with a deletion was missing 200 to 261 residues of the cps3D gene. Statistical analyses were done by the unpaired Student t test, which was used to determine the change in CT for each rough phase variant compared to that of the serotype 3 clone; P was <0.05 for all comparisons.

Reversion frequencies of seven rough phase variants. Daily subculturing of both the V11I and the I297T rough phase variants resulted in occasional spontaneous reversion to the mucoid phenotype. Sequencing of the cps3D promoter region and the coding sequences of the mucoid isolates revealed that the SNPs associated with the rough phenotype had reverted back to the wild type. Moreover, capsule ELISA of a reverted strain for each type of rough phase variant demonstrated that the reverted strain had >97% the parental amount of capsule. The reversion frequency for the V11I rough phase variant was 3.6 x 10^–5.

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Biofilms are present in the middle ear during recurrent otitis media and otitis media with effusion (13, 17). The serotype 3 strain that was used to form biofilms in this study was a representative strain of the serotype 3 clone that is associated with an increase in the proportion of cases of AOM in the PCV7 era (24). In this study, we found that nearly one-fourth of the strains visualized from 4- to 7-day-old biofilms had a rough morphology. The in vitro serotype 3 phase variants had a diverse array of mutations and included strains with point mutations and deletions in the first gene of the capsular operon, cps3D. The latter type of capsular rough phase variant has not been described previously. Unlike the nasopharynx, where at least 20% of the parental amount of capsule is required for colonization (21), in vitro biofilm-associated phase variants which generated <1% of the parental amounts of capsule and which had mutations in the cps3D gene were frequently detected. Spontaneous reversion to the parental phenotype and genotype was noted for two rough phase variants with missense mutations, suggesting that at least some of the biofilm-associated phase variants can transition between phenotypes.

The regulation of the capsule appears to occur at both the transcriptional level and the posttranscriptional level, as shown in Fig. 1. The capsule from the variant with the promoter down-mutation appeared to be regulated at the transcriptional level. In contrast, the remainder of the rough variants appeared to be regulated at the posttranscriptional level, since the higher cps3DSUM transcript levels did not result in more capsule compared to the amount for the serotype 3 clone. These missense and nonsense mutations in the cps3D gene likely result in defective and truncated proteins, respectively. These data suggest that the phase variants present in the middle ear during AOM likely contain subpopulations of pneumococci different from those observed in the nasopharynx during colonization.

The molecular mechanisms of phase variation are not well understood. Since phase variants can arise through point mutations (12), it is plausible that phase variation is partially regulated by mismatch repair genes. In S. pneumoniae, the mismatch repair genes include the Hex genes (11, 15) and the Hex-independent genes, the mutY and mutX genes (8, 25, 30), and the pms gene (22). The rough phase variants with deletions, which have not previously been described in a biofilm model, did not appear to arise through recombinational events of nontandem repeats (28). Instead, they may have arisen through a topoisomerase I-mediated process (6). In vitro biofilm-associated acapsular phase variants with tandem sequence duplications in cps3D have also been described in the literature (33) and may arise through either recA-independent or recA-dependent mechanisms, or through both types of mechanisms (27, 31, 32).

The main limitation of this work is the use of an in vitro rather than an in vivo biofilm model. The latter is needed to confirm the presence of rough variants within the middle ear biofilms generated during chronic otitis media and to determine the contribution of both the mucoid and the rough variants to the biofilm. The rough variants may arise to enhance biofilm formation. Akin to the cell walls of the transparent variants detected in the nasopharynx during colonization, these rough variants likely have thick cell walls with subcapsular molecules which promote attachment (7). In summary, these in vitro data indicate that biofilm-associated rough phase variants have a vast array of mutations within the cps3D gene and can regulate capsule production at multiple levels. Remarkably, these in vitro biofilm-associated phase variants do not have sufficient amounts of capsule to colonize the nasopharynx. Taken together, these data underscore the plasticity of the pneumococcus and its ability to rapidly adapt to the host environment.

 
We gratefully acknowledge Kelly Cole, Moon Nahm, and Jigui Yu for their excellent assistance with the ELISAs. We thank Eowyn Tinsley and Ivan Martinez for their technical expertise and Ellen Wald for providing the serotype 3 clone.

This work was supported in part by a NIH career development award (award K23 AI01788) to M. C. McEllistrem.

The authors do not have any conflict of interests with this paper.

 
* Corresponding author. Mailing address: Division of Infectious Diseases, University of Pittsburgh, Falk Medical Building, Suite 3A, 3601 Fifth Avenue, Pittsburgh, PA 15213-2582. Phone: (412) 648-6401. Fax: (412) 648-6399. E-mail: mcellistremc{at}dom.pitt.edu.

Published ahead of print on 8 November 2006.

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Journal of Clinical Microbiology, January 2007, p. 97-101, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01658-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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• Kurola, P., Tapiainen, T., Kaijalainen, T., Uhari, M., Saukkoriipi, A. (2009). Xylitol and capsular gene expression in Streptococcus pneumoniae. J Med Microbiol 58: 1470-1473 [Abstract] [Full Text]  
• Munoz-Elias, E. J., Marcano, J., Camilli, A. (2008). Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization. Infect. Immun. 76: 5049-5061 [Abstract] [Full Text]  
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