Closing the Gap: Harnessing next generation sequence technology to rapidly elucidate healthcare-associated infection transmission pathways Restricted; Files Only

Abstract

Each year, at least 2 million people acquire infections due to antibiotic resistant bacteria in the U.S., leading directly to at least 23,000 deaths (CDC, 2013). The spread of emerging antimicrobial resistance (AR) has increased the need to rapidly respond to outbreaks and improve infrastructures for detection and prevention of healthcare-associated infections (HAIs). Among the most common nosocomial infections, carbapenemase-resistant Enterobacteriaceae (CRE) pathogens cause over 9,000 HAIs per year, and are resistant to nearly all antibiotics including carbapenems – an antibiotic of last resort (The White House, 2014). Many carbapenems are harbored on mobile genetic elements (MGEs) and their transmission cannot be easily traced using traditional methods of molecular subtyping (e.g., pulsed-field gel electrophoresis, PFGE). Thus, advanced sequencing methods, such as whole-genome sequencing (WGS), are needed to rapidly identify additional transmission pathways and probable linked cases, beyond what PFGE provides. The purpose of this project was to implement WGS to elucidate the movement of MGEs harboring AR genes between multiple HAI bacterial species among high-risk patients residing in a LTACH facility in Orange County, Florida.

A variety of laboratory methods were performed to test 86 clinical and 91 environmental samples including culture and isolation, species identification, DNA extraction, and bacterial resistance mechanism testing. Combined with existing epidemiological data, PFGE was performed for cluster analyses and to identify possible common links between patients and their environment. WGS methods, by short and long-read sequencing platforms, were applied to provide increased resolution, confirm true relatedness, detect MGEs, and reveal plasmid-mediated gene(s). Bioinformatics tools were then applied to analyze raw data findings and identify specific AR characteristics associated with the targeted resistance gene, Klebsiella pneumoniae carbapenemase (blaKPC).

In both the clinical and environmental isolates, WGS identified multiple variants of KPC-producing genes harbored on multiple plasmids across multiple bacterial species in this single healthcare facility. These granular connections were not identified by PFGE. Plasmid discovery within this outbreak elucidated and confirmed the ability of MGEs to spread intra- and inter-organism, and persist within a healthcare environment, ultimately leading to patient acquisition. These findings support the hypothesis that MGEs, especially those containing carbapenemase genes, can be significant drivers of antibiotic resistance and HAI outbreaks.

Table of Contents

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER

           1   INTRODUCTION......................................................................................................... 1

                       1.1 BACKGROUND............................................................................................... 1

                       1.2 CURRENT GAPS............................................................................................. 5

                       1.3 PURPOSE……….............................................................................................. 5

                       1.4 SIGNIFCANCE................................................................................................. 6

           2   REVIEW OF THE LITERATURE............................................................................... 8

                       2.1 HEALTHCARE-ASSOCIATED INFECTIONS.............................................. 8

                      2.2 LONG-TERM ACUTE CARE HOSPITALS................................................... 8

                       2.3 ANTIBIOTIC RESISTANCE......................................................................... 10

                       2.4 HEALTHCARE ENVIRONMENT AND TRANSMISSION........................ 11

                       2.5 MOLECULAR AND GENOMIC TYPING................................................... 12

           3   APPROACH AND METHODS.................................................................................. 17

                       3.1 HYPOTHESIS................................................................................................. 17

                       3.2 INTRODUCTION........................................................................................... 17

                       3.3 POPULATION AND SAMPLE COLLECTION........................................... 19

                       3.4 RESEARCH DESIGN..................................................................................... 22

                       3.5 MATERIALS AND METHODS …………………………………………..26

           4   RESULTS..........................................................................................................36

                       4.1 OUTBREAK SUMMARY.............................................................................. 36

                       4.2 PFGE FINDINGS............................................................................................ 37

                       4.3 ISOLATE RELATEDNESS........................................................................... 37

                       4.4 GENOTYPIC ANTIBIOTIC RESISTANCE................................................. 40

                       4.5 MOBILE GENETIC ELEMENTS.................................................................. 40

                       4.6 TREE COMPARISONS.................................................................................. 42

           5   SUMMARY AND CONCLUSION............................................................................44

                       5.1 Summary of findings....................................................................................... 44

                       5.2 Conclusion....................................................................................................... 46

                       5.3 Lessons Learned.............................................................................................. 47

                       5.4 Strengths and Weaknesses............................................................................... 47

                       5.5 Next Steps for the Future................................................................................. 48

           REFERENCES.................................................................................................................. 60

List of Tables                                                                                                                                                    

Table 1: Bacterial species isolates for each patient sample........................................................... 20

Table 2: Overview of environmental sample collections.............................................................. 21

Table 3: Bacterial species isolates from environmental samples.................................................. 21

Table 4: The subset of blaKPC isolates selected for WGS testing.................................................. 24

Table 5: Isolate Relatedness - hqSNP matrix: Enterobacter (all isolates).................................... 53

Table 6: Isolate Relatedness - hqSNP matrix: Enterobacter roggenkampii................................. 53

Table 7: Isolate Relatedness - hqSNP matrix: Enterobacter hormaechei (ST 597)..................... 53

Table 8: Isolate Relatedness - hqSNP matrix: Klebsiella pneumoniae (all isolates).................... 54

Table 9: Isolate Relatedness - hqSNP matrix: Klebsiella pneumoniae (ST 14)............................ 55

Table 10: Isolate Relatedness - hqSNP matrix: Citrobacter isolates............................................ 55

Table 11: Isolate Relatedness - hqSNP matrix: Serratia isolates.................................................. 55

Table 12: Overview of MLST schemes and key AMR genes....................................................... 56

Table 13: PacBio genome hybrid assembly of the ~50Kb blaKPC-3 plasmid................................ 58

Table 14: PacBio genome hybrid assembly of the ~180Kb blaKPC-2 plasmid.............................. 59

Table 15: Phylogenetic tree landscapes – Enterobacter cloacae complex isolates...................... 43

Table 16: Phylogenetic tree landscapes – Klebsiella pneumoniae complex isolates.................... 43

List of Figures

Figure 1: Rationale of the statistical approach used in treespace.................................................. 25

Figure 2: Workflow of the QuAISAR-H pipeline......................................................................... 34

Figure 3: PacBio genomic comparison of the blaKPC-3 plasmid................................................... 41

Figure 4: PacBio genomic comparison of the blaKPC-2 plasmid................................................... 42

Figure 5: Enterobacter PFGE similarity tree structure................................................................. 50

Figure 6: Enterobacter hqSNP similarity tree structure................................................................ 50

Figure 7: Klebsiella PFGE similarity tree structure...................................................................... 51

Figure 8: Klebsiella hqSNP similarity tree structure..................................................................... 51

Figure 9: PFGE Dendrogram - Enterobacter cloacae complex isolates....................................... 52

Figure 10: PFGE Dendrogram - Klebsiella pneumoniae isolates................................................. 52

List of Abbreviations

AMR                         Antimicrobial resistance

AR                            Antibiotic resistance

CPO                          Carbapenemase-producing organisms

CRE                          Carbapenemase-resistant Enterobacteriaceae

DNA                         Deoxyribonucleic acid

GN                            Gram-negative bacteria

HAI                           Healthcare-associated infections

ICU                           Intensive care unit

PPS                           Point-prevalence surveys

PFGE                        Pulsed-field gel electrophoresis

LTACH                     Long-term acute care hospitals

KPC                          Klebsiella pneumoniae carbapenemase

MALDI-TOF           Matrix-Assisted Laser Desorption Ionization Time-of-Flight

MGE                        Mobile genetic elements

MLST                      Multilocus sequence typing

NGS                         Next Generation Sequencing

RT-PCR                   Real-Time Polymerase Chain Reaction

SNP                          Single nucleotide polymorphisms

SNV                         Single nucleotide variants

WGS                        Whole Genome Sequencing

VIM                         Verona Integron-Encoded Metallo-β-Lactamase

CRPA                       Carbapenem-Resistant Pseudomonas aeruginosa

About this Master's Thesis

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School	Rollins School of Public Health
Department	Executive Masters of Public Health - MPH
Degree	M.P.H.
Submission	Master's Thesis
Language	English
Research Field	Health Sciences, Epidemiology Environmental Sciences Biology, Microbiology
Keyword	Infection Control and Prevention
Committee Chair / Thesis Advisor	Lesley McGee, Emory University
Committee Members	Alison Laufer Halpin, CDC

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