Horizontal Transfer of Antibiotic Resistance in Streptococcus pneumoniae Restricted; Files Only
Antezana, Brenda (Summer 2023)
Abstract
Streptococcus pneumoniae (pneumococcus) is a nasopharyngeal commensal that may spread to local or sterile sites, causing non-invasive or invasive pneumococcal disease. Antibiotic resistance was first observed in S. pneumoniae during the 1960s against penicillin and expanded to macrolides in the 1990s. One key contributor to macrolide resistance is Tn916-related integrative and conjugative elements (ICEs), such as Tn2009 (23.5 kb), Tn6002 (20.8 kb), and Tn2010 (26.3 kb). Unlike in vitro planktonic cell transformation, efficient Tn916-related ICE transfer was observed in dual-strain biofilms formed on human nasopharyngeal cells. Investigation of the transfer mechanism disproved the involvement of conjugation based on limited conjugative gene expression, absence of ICE circular intermediates, and no change in ICE transfer frequency with a conjugative mutant donor. However, competence and transformation mutant studies, extracellular DNA removal by DNase I treatment, and recombinant genome analyses supported transformation and homologous recombination as the major mechanism for pneumococcal Tn916- related ICE dissemination. Contribution of Tn916-related ICEs to pneumococcal macrolide resistance and effects of pneumococcal conjugate vaccines (PCVs) to ICE circulation were explored in the United States using genomes of 4,560 PubMLST S. pneumoniae isolates collected during 1916-2021. In this collection, Tn916-related ICE frequency increased over time. This rise was associated with a decrease in ICE+ isolates of vaccine serotypes upon PCV introductions, but a marked increase in ICE+ isolates of non-vaccine serotypes, such as 15A and 23A, which are not included in the current PCV15 and PCV20. Tn916-related ICE macrolide resistance shifted from Tn2009 mefE/mel-mediated macrolide efflux to ermB-mediated ribosomal methylation of Tn6002 and Tn2010 by the 2010s. Transformation of DNA associated with naturally released pneumococcal extracellular vesicles (EVs) was evaluated under planktonic and biofilm conditions and was not more efficient compared to free DNA. While in vitro conditions enabled EV transformation of streptomycin (rpsL/K56T) and erythromycin (738-bp ermB) resistance, biofilms facilitated natural EV transformation of the 5.4-kb Mega element and Tn2009. SNP analyses of biofilm EV recombinants revealed homologous recombination of ~9-26 kb DNA fragments. This work contributes new mechanistic and population findings of antibiotic resistance dissemination in S. pneumoniae, highlighting the importance of transformation, biofilms, and continuing pneumococcal evolution.
Table of Contents
Chapter 1: Introduction to Streptococcus pneumoniae 1
Chapter 2: Dissemination of Tn916-related integrative and conjugative elements in Streptococcus pneumoniae occurs by transformation and homologous recombination in nasopharyngeal biofilms 30
Abstract 31
Importance 32
Introduction 33
Results 34
Discussion 46
Materials and Methods 50
Acknowledgements 60
Figures and Tables 61
Supplemental Figures and Tables 70
References 82
Chapter 3: Streptococcus pneumoniae Tn916-related integrative and conjugative elements in
the United States in the pre- and post-pneumococcal conjugate vaccine eras (1916-2021) 95
Abstract 96
Importance 97
Introduction 98
Results 100
Discussion 104
Materials and Methods 110
Acknowledgements 112
Figures 113
Supplemental Figures 118
References 119
Chapter 4: Extracellular vesicles and transformation of antimicrobial resistance genetic determinants in Streptococcus pneumoniae 132
Abstract 133
Importance 134
Introduction 135
Results 137
Discussion 143
Materials and Methods 147
Acknowledgements 151
Figures and Tables 153
Supplemental Tables 158
References 159
Chapter 5: Conclusions and future directions 168
Appendix A: Interaction between Streptococcus pneumoniae and Staphylococcus aureus Generates •OH Radicals That Rapidly Kill Staphylococcus aureus Strains 191
Appendix B: Lactoferrin Disaggregates Pneumococcal Biofilms and Inhibits Acquisition of Resistance Through Its DNase Activity 210
Chapter 2: Dissemination of Tn916-related integrative and conjugative elements in Streptococcus pneumoniae occurs by transformation and homologous recombination in nasopharyngeal biofilms 30
Figure 1. Efficient transfer of pneumococcal Tn916-related ICEs occurs in dual-strain nasopharyngeal biofilms. 61
Figure 2. Sequence alignments of prototype Tn916 with pneumococcal Tn916-related ICEs. 62
Figure 3. Pneumococcal Tn2009 does not form circular intermediates (CIs) nor transfer by conjugation. 63
Figure 4. Pneumococcal Tn2009 transfer in human nasopharyngeal biofilms requires competence and transformation machinery in the recipient. 64
Figure 5. Partial pneumococcal Tn6002 lacking conjugative regulatory mechanism transfers via transformation and homologous recombination in a strain-dependent manner. 66
Figure 6. Whole-genome sequencing reveals homologous recombination of large Tn2009- containing fragments (~33 to ~55 kb) from GA16833 into D39 bioreactor recombinants. 67
Figure 7. Efficient transfer of Tn916-related ICEs via transformation and homologous recombination in nasopharyngeal biofilms occurs in other S. pneumoniae strains. 68
Table 1. Tetracycline-induced fold change in tetM and conjugative gene expression of Tn916 and Tn2009. 69
Table 2. Homologous recombination of donor DNA fragments of various sizes with intact Tn2009 into D39 bioreactor recombinant genomes. 69
Table 3. Early com gene expression in recipient D39Ery/Str from dual-strain bioreactor biofilm with BASP8 compared to in vitro transformation without CSP addition. 69
Figure S1. Similar extracellular DNA concentrations are secreted from competence-deficient mutant recipient strain BASP2 and Tn2009 ICE donor GA16833 in nasopharyngeal dual-strain biofilms. 70
Figure S2. Single recipient strain biofilm transforms point mutation-mediated resistance but not Tn916-related ICE resistance. 71
Figure S3. In vitro transformation at 35°C and 37°C facilitates recipient uptake of point mutation- mediated resistance but not Tn916-related ICE resistance. 72
Table S1. Strains used in this study. 73
Table S2. Primers and probes used in this study. 75
Table S3. Genetic identity and rF data for D39Str/Tmp and GA16833Tet/Ery or GA47281Tet/Ery bioreactor co-inoculation strains and recombinants. 79
Table S4. Homologous recombination of variably sized donor DNA fragments with intact ICE into bioreactor recombinant genomes. 80
Chapter 3: Streptococcus pneumoniae Tn916-related integrative and conjugative elements in the United States in the pre- and post-pneumococcal conjugate vaccine eras (1916-2021) 95
Figure 1. Tn916-related ICE frequency has increased in US S. pneumoniae isolates. 113
Figure 2. Shift in the distribution of Tn916-related ICEs in the US during pre-PCV7, PCV7, and PCV13 eras. 114
Figure 3. Tn916-related ICE-containing S. pneumoniae isolates from 1916 to 2021 in the US. 115
Figure 4. Tn916-related ICEs emerged in non-vaccine serotypes after PCV7 and PCV13 introductions in the US. 116
Figure 5. Serotype-specific analysis for Tn916-related ICE-containing isolates in the US, 1995- 2021. 117
Figure S1. S. pneumoniae isolates carrying Tn916-related ICEs in US, Thailand, and South Africa during pre-PCV7 and post-PCV7/13 eras. 118
Chapter 4: Extracellular vesicles and transformation of antimicrobial resistance genetic determinants in Streptococcus pneumoniae 132
Figure 1. Transmission electron microscopy (TEM) images of S. pneumoniae extracellular vesicles. 153
Figure 2. S. pneumoniae extracellular vesicles transform externally associated DNA into planktonic cells or pneumococcal biofilms at reduced frequencies compared to genomic DNA transformation. 154
Figure 3. EV-mediated transformation of larger antimicrobial resistance determinant occurs in pneumococcal biofilms as opposed to planktonic cells though at reduced frequencies compared to genomic DNA transformation. 155
Figure 4. EV-mediated transformation of pneumococcal biofilms occurs in presence or absence of synthetic competence stimulating peptide. 156
Table 1. Quantification of S. pneumoniae extracellular vesicle (EV) protein and DNA content. 157
Table 2. Integration of donor DNA fragments delivered by S. pneumoniae extracellular vesicles occurs via homologous recombination. 157
Table S1. Strains used in this study. 158
Table S2. Primers used in this study. 158
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