D-068. Isolation and Characterization of Streptococcus pneumoniae Biofilm Mutants in Vivo

E. J. Muñoz-Elías1, J. Marcano2, A. Camilli1;
1Howard Hughes Med. Inst. and Tufts Univ., Boston, MA, 2Univ. of Puerto Rico, Mayagüez, PR.

Asymptomatic colonization of the human nasopharynx by S. pneumoniae -a primary etiological agent of otitis media, pneumonia, and meningitis- is common and positively correlates with the incidence of pneumococcal disease and antibiotic resistance. Yet, pneumococcal factors involved in colonization remain poorly understood. Many bacterial infections are thought to involve surface-adherent biofilms whose establishment on host tissues is intrinsically linked to adaptations which protect biofilm bacteria from host defenses and antibiotics. To gain insight into the genetics of biofilm formation by S. pneumoniae (clinical isolate TIGR4), we conducted an in vitro screen for biofilm defective mutants. In a screen of 6,000 mariner transposon isogenic mutants, we repeatedly and almost exclusively isolated biofilm overproducing acapsular mutants resulting from insertions in the capsule locus, suggesting the capsule was antagonistic to biofilm formation. Consequently, we generated 7,000 additional transposon mutants in an S. pneumoniae acapsular mutant background and conducted a second screen. Following this approach, we isolated 73 insertions in 43 different genes and 7 promoters. The collection of mutants includes genes encoding bona fide and putative choline binding proteins, adhesins, synthases of membrane and cell wall components, extracellular and cell wall proteases, efflux pumps, ABC and PTS transporters, transcriptional regulators, as well as several hypothetical ORFs. Interestingly, while five insertions mapped to rrgA, encoding a subunit of a recently described surface pilus, mutants of rrgB and rrgC -encoding the other two pilus subunits- had no biofilm defects, implicating the RrgA adhesin but not the pilus structure per se in biofilm formation. To correlate our findings to host infection, we transferred the mutations to the wildtype encapsulated parental strain and then tested the fitness of 26 of these mutants in vivo. Strikingly, we found that 20 of these mutants were highly impaired for colonization of the murine nasopharynx, thus establishing a link between biofilm formation and colonization of the mammalian host.