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

• Type of Material: Article
• Authors: Santiago M. Lattar, Emory University; Xueqing Wu, Emory University; Jennifer Brophy, Emory University; Fuminori Sakai, Emory University; Keith P Klugman, Emory University; Jorge Vidal Graniel, Emory University
• Language: English
• Date: 2018-05-01
• Publisher: American Society for Microbiology: Open Access Journals
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2018 Lattar et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1128/mBio.00561-18
• Title of Journal or Parent Work: mBio
• ISSN: 2150-7511
• Volume: 9
• Issue: 3
• Grant/Funding Information: Confocal studies were supported in part by funds from the Integrated Cellular Imaging (ICI) pediatric core and the Emory+Children’s Pediatric Research Center to J.E.V.; This study was supported by a grant from the National Institutes of Health (R21AI112768-01A1 to J.E.V.).
• Keywords: unidirectional transformation; consortial biofilms; CONJUGATE VACCINE; STRAIN D39; Streptococcus pneumoniae; STREPTOCOCCUS-PNEUMONIAE; VIBRIO-CHOLERAE; antibiotic resistance; DNA-UPTAKE; SECRETION SYSTEM; UNITED-STATES; Science & Technology; MULTIPLEX PCR; GENOME SEQUENCE; Life Sciences & Biomedicine; Microbiology; GENETIC-TRANSFORMATION
• Research Categories: Health Sciences, Public Health; Health Sciences, Medicine and Surgery

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 S2Tetand S4Strin a bioreactor simulating the human nasopharynx led to the generation of SpnTet/Strrecombinants. Double-resistant pneumococci emerged soon after 4 h postinoculation at a recombination frequency (rF) of 2.5 × 10-4while 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., S4Strand S19FTmp) were incubated together, leading to S19FStr/Tmpbut 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. IMPORTANCE Pneumococcal transformation in the human nasopharynx may lead to the acquisition of antibiotic resistance genes or genes encoding new capsular variants. Antibiotics and vaccines are currently putting pressure on a number of strains, leading to an increase in antibiotic resistance and serotype replacement. These pneumococcal strains are also acquiring virulence traits from vaccine types via transformation. In this study, we recapitulated multiple-strain colonization with strains carrying a resistance marker and selected for those acquiring resistance to two or three antibiotics, such as would occur in the human nasopharynx. Strains acquiring dual and triple resistance originated from one progenitor, demonstrating that transformation was unidirectional. Unidirectional transformation was the result of inhibition of transformation of donor strains. Unidirectional transformation has implications for the understanding of acquisition patterns of resistance determinants or capsule-switching events.