High levels of genetic recombination during nasopharyngeal carriage and biofilm formation in Streptococcus pneumoniae

Abstract

Transformation of genetic material between bacteria was first observed in the 1920s using Streptococcus pneumoniae as a model organism. Since then, the mechanism of competence induction and transformation has been well characterized, mainly using planktonic bacteria or septic infection models. However, epidemiological evidence suggests that genetic exchange occurs primarily during pneumococcal nasopharyngeal carriage, which we have recently shown is associated with biofilm growth, and is associated with cocolonization with multiple strains. However, no studies to date have comprehensively investigated genetic exchange during cocolonization in vitro and in vivo or the role of the nasopharyngeal environment in these processes. In this study, we show that genetic exchange during dual-strain carriage in vivo is extremely efficient (10(-2)) and approximately 10,000,000-fold higher than that measured during septic infection (10(-9)). This high transformation efficiency was associated with environmental conditions exclusive to the nasopharynx, including the lower temperature of the nasopharynx (32 to 34°C), limited nutrient availability, and interactions with epithelial cells, which were modeled in a novel biofilm model in vitro that showed similarly high transformation efficiencies. The nasopharyngeal environmental factors, combined, were critical for biofilm formation and induced constitutive upregulation of competence genes and downregulation of capsule that promoted transformation. In addition, we show that dual-strain carriage in vivo and biofilms formed in vitro can be transformed during colonization to increase their pneumococcal fitness and also, importantly, that bacteria with lower colonization ability can be protected by strains with higher colonization efficiency, a process unrelated to genetic exchange.

Importance: Although genetic exchange between pneumococcal strains is known to occur primarily during colonization of the nasopharynx and colonization is associated with biofilm growth, this is the first study to comprehensively investigate transformation in this environment and to analyze the role of environmental and bacterial factors in this process. We show that transformation efficiency during cocolonization by multiple strains is very high (around 10(-2)). Furthermore, we provide novel evidence that specific aspects of the nasopharyngeal environment, including lower temperature, limited nutrient availability, and epithelial cell interaction, are critical for optimal biofilm formation and transformation efficiency and result in bacterial protein expression changes that promote transformation and fitness of colonization-deficient strains. The results suggest that cocolonization in biofilm communities may have important clinical consequences by facilitating the spread of antibiotic resistance and enabling serotype switching and vaccine escape as well as protecting and retaining poorly colonizing strains in the pneumococcal strain pool.

FIG 1

Efficiency of transformation of antibiotic resistance elements between SP670 (Pen^r) and D39-C08P2 (Erm^r) during nasopharyngeal cocolonization, sequential cocolonization, and sepsis.

FIG 2

(A) SP670 transformation efficiency during biofilm or planktonic growth after addition of D39-C08P2 natural lysate. (B) Transformation efficiency of dual-strain biofilms. Equal inocula of each strain were cocultured in biofilms on top of prefixed epithelial substrata for 72 h before being plated on selective medium: D39-C08P2/SP670, HT6/SP670, JD908/SP670, and SP456/HT6 were all grown on Pen^r-plus-Erm^r plates, whereas STM68/D39-C08P2 were grown on Cm^r-plus-Erm^r plates.

FIG 3

(A) Expression of competence genes during biofilm formation. Levels of gene expression were quantitated by qRT-PCR using the standard curve method and normalized to levels of 16S rRNA as an internal control. All data are reported as fold change in gene expression compared to a mid-logarithmic-phase culture of D39 (for D39 biofilms) or Rx1 (for Rx1 cultures). (B) Transformation efficiency of dual-strain biofilms of SP670 and D39-C08P2 over 72 h. (C) Transformation efficiency of SP670 biofilms after addition of chromosomal D39-C08P2 DNA in chemically defined medium (CDM) or THY complex medium with or without the addition of competence-stimulating peptide (CSP). (D) Transformation efficiency of planktonic cultures of D39 and AM1000 using D39-C08P2 chromosomal DNA compared to transformation efficiency of single-strain biofilms formed by D39 and AM1000, inoculated with D39-C08P2 chromosomal DNA. Transformation efficiency was measured as the CFU/biofilm carrying both Pen^r and Erm^r as a ratio of the total biomass in the biofilm (B and C) or CFU of the total biomass carrying Erm^r (D).

FIG 4

(A) Transformation efficiency of dual-strain biofilms cocultured on an epithelial substratum or on glass for 72 h. (B) Transformation efficiency of dual-strain biofilms SP670/D39-C08P2 and HT6/SP670 or single-strain biofilms SP670 and Rx1 inoculated with D39-C08P2 chromosomal DNA at 34°C and 37°C for 72 h.

FIG 5

(A to C) Total population and subpopulations from dual-strain biofilms of HT6/SP670 (A), SP670/JD908 (B), and P2A1/SP670 (C), compared to the biomass of single-strain biofilms. (D) Total nasopharyngeal colonization and subpopulations after 48-h dual-strain colonization by SP670 and D39-C08P2 compared to colonization by each single strain. (E) Total bacterial burden and isolated subpopulations during sepsis induced by SP670 and D39-C08P2. (F) Isolated encapsulated (EF3030) and unencapsulated (AM1000) populations recovered after 48 h of nasopharyngeal carriage from dual-strain challenge (lanes 1 and 2, coculture of AM1000 and EF3030) or single-strain nasopharyngeal carriage (lanes 3 and 4).

FIG 6

Colonization levels of each subpopulation 48 h after dual-strain colonization between D39 and TRE121 (lanes 1 to 4) and single-strain colonizations (lanes 5 to 7). Lane 1, total recovered pneumococci; lane 2, pspA/pspC null pneumococcus TRE121; lane 3, TRE121 pneumococci with repaired pspC gene; lane 4, TRE121 pneumococci with repaired pspA gene; lane 5, single-strain colonizing ability of TRE121 alone; lane 6, single-strain colonizing ability of D39 alone; lane 7, rechallenge of mice with isolate from dual colonization with strain with repaired pspC gene (from lane 3).