Colistin Heteroresistance: An Under-recognized and Challenging Form of Antibiotic Resistance Open Access

Abstract

Antibiotic resistance threatens the delivery of safe and effective healthcare resulting in 2 million infections and 23,000 deaths annually in the United States. Further complicating this epidemic are unexplained antibiotic treatment failures caused by bacteria that appear susceptible to an antibiotic. The phenomenon of heteroresistance occurs when a minor resistant subpopulation exists within a majority susceptible strain. We describe here several instances of heteroresistance to the last-line peptide antibiotic colistin. First, we observed colistin heteroresistant isolates of Enterobacter cloacae that were able to mediate lethal infection during colistin treatment. Interestingly, we observed that the resistant subpopulation was augmented by drug treatment as well as host immune pressure, through macrophages and host antimicrobial compounds. We additionally show that these heteroresistant strains can be misclassified as susceptible by routine susceptibility testing. This could conceivably lead to incorrect treatment with colistin which may lead to unexplained treatment failure. We additionally chacterized strains of colistin heteroresistant Klebsiella pneumoniae, which were additionally resistant to the last line carbapenems. Thus, these isolates would rely on a last line drug such as colistin and could also lead to colistin treatment failure. To assess the extent of heteroresistance in clinical isolates, we conducted a study of heteroresistance in a wide-ranging pool of carbapenem resistant Enterobacteriaceae in the United States. We observed a rate of heteroresistance of over 10% in these isolates. Over 90% of these isolates were misclassified as colistin susceptible, thus underestimating total colistin non-susceptibility by over 2-fold. These findings highlight a largely unappreciated phenomenon that could have a significant impact upon antibiotic treatment outcome, exacerbating the menace of antibiotic resistance.

Table of Contents

Chapter 1: Introduction ............................................................................................................... 1

History of Antibiotic Therapy..................................................................................................... 1

Antibiotic Resistance................................................................................................................... 2

Polymyxins and antimicrobial peptide resistance ....................................................................... 4

Figure 1.1. Bacterial surface modifications that enhance CAMP resistance........................... 6

Antibiotic Heteroresistance....................................................................................................... 24

Figure 1.2. Characteristics of heteroresistance to antibiotics suggest possible clinical impact ...............27

Chapter 2: Antibiotic failure mediated by resistant subpopulation in Enterobacter cloacae ........30

Abstract ........................................................ 31

Results ...........................................................31

Figure 2.1. A colistin resistant subpopulation increases in frequency during in vivo infection ........ 33

Figure 2.2. Innate immune host defenses are required for the increased frequency of the colistin resistant subpopulation during infection ...35

Figure 2.3. R/S is refractory to colistin during infection and leads to colistin treatment failure.......... 38

Figure 2.4. PhoQ is required for the colistin resistant subpopulation and treatment failure....41

Figure 5. Clinical isolate harboring an undetected colistin resistant subpopulation causes a lethal, antibiotic resistant infection..... 44

Discussion ................................................................................................ 44

Methods..................................................................................................... 46

Supplemental Figures................................................................................. 54

Figure S2.1. Etests of colistin susceptible and resistant isolates .............. 54

Figure S2.2. Bacteria from high and low antibiotic growth conditions behave identically after passage ......... 55

Figure S2.3. DNA sequencing of susceptible and resistant subpopulations ..... 56

Figure S2.4. Increase in the frequency of the colistin resistant subpopulation in the liver during in vivo infection .........57

Figure S2.5. Frequency of the colistin resistant subpopulation increases during in vivo infection.............57

Figure S2.6. Macrophage depletion via clodronate liposomes.......................... 58

Figure S2.7. The human antimicrobial peptide LL-37 leads to an increase in frequency of the colistin resistant subpopulation .. 58

Figure S2.8. Increased CFU during infection of mice lacking host antimicrobials .............. 59

Figure S2.9. Infection of mice lacking individual host antimicrobials ................................. 60

Figure S2.10. In vivo growth and expansion of R/S during colistin treatment of mice ........ 61

Figure S2.11. Expression of PhoQ regulated lipid A modification genes is induced by colistin ......... 62

Figure S2.12. Lipid A analysis reveals modifications present in resistant subpopulation .... 63

Figure S2.13. Kanamycin persisters in R/S are not dependent on PhoQ .............................. 64

Figure S2.14. R/S deficient in PhoQ lacks ability to induce colistin resistant subpopulation .......65

Figure S2.15. The frequency of the colistin resistant subpopulation of R/S-lo increases in the presence of drug....... 66

Figure S2.16. Colistin selects for the colistin resistant subpopulation of R/S-lo .................. 66

Figure S2.17. Host antimicrobials lead to an increase in the frequency of the colistin resistant subpopulation of R/S-lo ...... 67

Figure S2.18. The frequency of the R/S-lo colistin resistant subpopulation increases in macrophages ........68

Figure S2.19. The frequency of the R/S-lo resistant subpopulation increases during mouse infection.......68

Figure S2.20. Macrophages are required for the increase in the frequency of the R/S-lo resistant subpopulation during infection...... 69

Figure S2.21. Specific host antimicrobials contribute to the increased frequency of the R/Slo subpopulation in vivo...........69

Figure S2.22. Inefficacy of colistin in reducing the levels of strain R/S-lo during in vivo infection.....70

Figure S2.23. Schematic indicating how antibiotic-resistant subpopulations can lead to unexplained clinical treatment failure ....... 71

Chapter 3: Carbapenem-resistant Klebsiella pneumoniae exhibiting clinically undetected colistin heteroresistance lead to treatment failure in a murine model of infection. .... 72

Abstract ..............................................73

Introduction .......................................74

Results ................................................74

Figure 3.1. Colistin heteroresistant, carbapenem-resistant Klebsiella pneumoniae harbor clinically undetected resistant subpopulations ....76

Figure 3.2. K. pneumoniae isolates lead to in vivo colistin treatment failure ....................... 79

Discussion ......................................... 80

Supplemental Figures........................ 82

Table S3.1. Antibiograms of colistin heteroresistant K. pneumoniae isolates ...................... 82

Table S3.2. Beta-lactam resistance genes in K. pneumoniae isolates ................................... 83

Figure S3.2. Frequency of the resistant subpopulation increases in the presence of colistin ...83

Figure S3.1. Increased broth microdilution incubation time facilitates detection of colistin heteroresistance ....................84

Figure S3.3. Workflow for genomic and transcriptomic analysis of colistin susceptible and resistant subpopulations..... 85

Figure S3.4. Expression of PhoPQ pathway genes in susceptible and resistant subpopulations.......... 86

Figure S3.5. Frequency of the resistant subpopulation increases during in vivo infection ... 87

Chapter 4 Heteroresistance to colistin among carbapenem-resistant Enterobacteriaceae: A Multistate Study, 2012-2015.... 88

Abstract ......................... 89

Introduction .........90

Results ................. 90

Table 4.1 Sex, Age, and Culture Source of Carbapenem Resistant Enterobacteriaceae....... 91

Table 4.2 Genus and State of Origin of Carbapenem Resistant Enterobacteriaceae............. 93

Figure 4.1 Rate of Colistin Heteroresistance by Genus, (2012-2015)................................... 94

Table 4.4 State of Origin of Carbapenem Resistant Klebsiella Species................................ 97

Table 4.5 Colistin Susceptibility Results of Enterobacteriaceae by Clinical and Laboratory Testing ...99

Discussion ..............................................................................................................100 Methods..................................................................................................................103

Supplemental Figures............................................................................................ 107

Table S1. Susceptibility to Last-line Antibiotics by Clinical Testing ...................... 107

Figure S1. Rate of Enterobacter Species and Heteroresistance by Year, 2012-2015.......... 107

Figure S2. Phylogenetic Tree, Resistance Genes, and Sequence Type of Colistin Heteroresistant Klebsiella Species...... 108

Chapter 5: Discussion and Relevance ............................................................. 109

Immune pressure drives antibiotic resistance.................................................110

Detection of heteroresistance .......................................................................111

Heteroresistance to other drugs ................................................................... 114

Multiple heteroresistance ...........................................................................116

Acknowledgements .....................................................................................118

References................................................................................................... 120

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Immunology and Molecular Pathogenesis
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Microbiology Health Sciences, Epidemiology Health Sciences, Immunology
Keyword	CRE colistin heteroresistance carbapenem resistant Enterobacteriaceae antibiotic resistance subpopulations
Committee Chair / Thesis Advisor	Weiss, David, Emory University
Committee Members	Tirouvanziam, Rabindra, Emory University Shafer, William, Emory University Neish, Andrew, Emory University Rengarajan, Jyothi, Emory University

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