A Mechanism of Unidirectional Transformation, Leading to Antibiotic Resistance, Occurs within Nasopharyngeal Pneumococcal Biofilm Consortia

Abstract

Streptococcus pneumoniae acquires genes for resistance to antibiotics such as streptomycin (Str) or trimethoprim (Tmp) by recombination via transformation of DNA released by other pneumococci and closely related species. Using naturally transformable pneumococci, including strain D39 serotype 2 (S2) and TIGR4 (S4), we studied whether pneumococcal nasopharyngeal transformation was symmetrical, asymmetrical, or unidirectional. Incubation of S2Tet and S4Str in a bioreactor simulating the human nasopharynx led to the generation of SpnTet/Str recombinants. Double-resistant pneumococci emerged soon after 4 h postinoculation at a recombination frequency (rF) of 2.5 × 10−4 while peaking after 8 h at a rF of 1.1 × 10−3. Acquisition of antibiotic resistance genes by transformation was confirmed by treatment with DNase I. A high-throughput serotyping method demonstrated that all double-resistant pneumococci belonged to one serotype lineage (S2Tet/Str) and therefore that unidirectional transformation had occurred. Neither heterolysis nor availability of DNA for transformation was a factor for unidirectional transformation given that the density of each strain and extracellular DNA (eDNA) released from both strains were similar. Unidirectional transformation occurred regardless of the antibiotic-resistant gene carried by donors or acquired by recipients and regardless of whether competence-stimulating peptide-receptor cross talk was allowed. Moreover, unidirectional transformation occurred when two donor strains (e.g., S4Str and S19FTmp) were incubated together, leading to S19FStr/Tmp but at a rF 3 orders of magnitude lower (4.9 × 10−6). We finally demonstrated that the mechanism leading to unidirectional transformation was due to inhibition of transformation of the donor by the recipient.

Puzzling Over the Pneumococcal Pangenome

“…While transformation frequencies vary across strains and conditions, transformation in pneumococcus can be highly efficient. In biofilm grown cells in a bioreactor, transformation reaches rates on the order of 10 -3 ( Lattar et al, 2018 ). It is noteworthy, that in these conditions recombination between strains may be unidirectional, although the mechanism behind this intriguing observation remains to be uncovered ( Lattar et al, 2018 ).…”

“…In biofilm grown cells in a bioreactor, transformation reaches rates on the order of 10 -3 ( Lattar et al, 2018 ). It is noteworthy, that in these conditions recombination between strains may be unidirectional, although the mechanism behind this intriguing observation remains to be uncovered ( Lattar et al, 2018 ). In animal models of dual-strain carriage, transformation reaches rates on the order of 10 -2 ( Marks et al, 2012b ).…”

High-Level Macrolide Resistance Due to the Mega Element [mef(E)/mel] in Streptococcus pneumoniae

“…This dual element macrolide resistance genotype results in an MLS B phenotype with high-level macrolide resistance. These elements are not only able to transmit between co-resident serotypes of S. pneumoniae within the nasopharyngeal niche, but also have the possibility of transmitting to other naturally competent bacteria present in the nasopharynx, such as the clinically relevant Haemophilus influenzae (Lattar et al, 2018).…”

Lactoferrin Disaggregates Pneumococcal Biofilms and Inhibits Acquisition of Resistance Through Its DNase Activity

“…To evaluate the effect of bLF on recombination frequency (rF), we followed the methodology established by Lattar et al (2018) with a small modification. Detroit 562 cells (ATCC CCL-138) were grown on snapwell filters (Corning, Corning, NY, United States) for about 4–5 days (until polarized).…”