Aminoglycoside Heteroresistance: Gene Amplification and Antibiotic Treatment Failure in Gram-Negative Pathogens Open Access

Sherman, Edgar (Fall 2020)

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Antibiotic resistance is a global challenge that threatens public healthcare by reducing our ability to prevent and treat bacterial infections. Antibiotic resistance results in approximately 2.8 million infections and 35,000 deaths annually in the United States. Further challenging our limited treatment strategies is unexplained antibiotic treatment failure, which may be due to heteroresistance. Heteroresistance occurs when a bacterial strain harbors a majority susceptible population as well as a minority resistant subpopulation. The resistant subpopulations can go undetected by clinical diagnostic techniques but nonetheless prevent antibiotic therapy from clearing an infection. In this report, we describe heteroresistance to aminoglycosides. Many Gram-negative pathogens have acquired widespread resistance to multiple classes of antibiotics but remain susceptible to aminoglycosides, leading to a revived interest in their clinical use. Here, we report results of a surveillance study for aminoglycoside heteroresistance in the Gram-negative pathogens carbapenem-resistant -Acinetobacter baumannii (CRAB) and -Enterobacteriaceae (CRE; Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae). We find that in sampling of multidrug resistant clinical isolates, heteroresistance was observed to the drugs tobramycin, gentamicin, and amikacin. Interestingly, we report the presence and amplification of an aminoglycoside modifying enzyme aadB in over one-third of the tobramycin heteroresistant CRAB isolates. Additionally, in CRE we compared our detection rate of aminoglycoside heteroresistance to clinical diagnostic data and report the frequency of inaccurate diagnostic testing. To explore the mechanisms facilitating heteroresistance, we use a genetic approach to analyze a tobramycin heteroresistant strain of E. cloacae, Mu1307. We find that Mu1307 exhibits amplification of the aminoglycoside modifying enzyme aadB. We describe amplifications that are found in distinct populations and that amplifications are unstable and reverted in the absence of the antibiotic. Furthermore, we show that tobramycin heteroresistance is mediated by aadB utilizing a knockout strain of Mu1307. Finally, we show that tobramycin heteroresistance causes treatment failure. These findings demonstrate the in vivo impact of aminoglycoside heteroresistance and highlight the clinical challenges that may be posed by this form of antibiotic resistance.

Table of Contents

Chapter 1: Introduction. 1

Antibiotic Resistance. 1

Aminoglycosides and Resistance Mechanisms. 3

Figure 1.1. Examples of Aminoglycoside Resistance Mechanisms. 5

Heteroresistance. 7

Homologous Recombination and Gene Amplification. 9

Chapter 2: Aminoglycoside Modifying Enzyme aadB in Heteroresistant Carbapenem-Resistant Acinetobacter baumannii Clinical isolates mediates treatment failure. 12

Abstract 13

Introduction. 14

Results. 16

Figure 2.1. Heteroresistance Incidence Rates in Carbapenem-Resistant Acinetobacter baumannii (CRAB). 18

Table 2.1. Clinical Summary of Vs. Rates of Heteroresistance in CRAB Isolates. 19

Table 2.3. MIC Values and PAP Results for Tobramycin Heteroresistant CRAB Isolates Encoding aadB. 22

Figure 2.2. Amplification of aadB in CRAB Clinical Isolates. 24

Figure 2.3. Increase in aadB Copy Number Correlates with Higher MIC Value in CRAB. 25

Figure 2.4. Tobramycin Heteroresistance Causes Tobramycin Treatment Failure in AB5075. 27

Figure 2.5. AadB Mediates Tobramycin Treatment Failure in Heteroresistant Isolate AB5075. 28

Discussion. 29

Materials and Methods. 31

Supplemental Material 35

Supplementary Figure 1. Aminoglycoside Population Analysis Profile Distribution Among CRAB Isolates. 38

Supplementary Figure 2. Population Analysis Profiles of aadB-positive CRAB Isolates. 39

Supplementary Figure 3. AadB is Required for Tobramycin Heteroresistance in A. baumannii AB5075. 40

Chapter 3: Antibiotic Treatment Failure and Gene Amplifications detected in Aminoglycoside Heteroresistant Enterobacter cloacae. 41

Abstract 42

Introduction. 43

Results. 46

Table 3.1. Aminoglycoside Heteroresistance and Undetected Heteroresistance Among CRE Isolates. 48

Figure 3.1. Rates of Undetected Heteroresistance Among Carbapenem-Resistant Enterobacteriaceae (CRE) Isolates. 49

Figure 3.2. aadB is Required for Tobramycin Resistance and the Presence of Resistant Subpopulations. 51

Figure 3.3. Amplification of Resistance Genes in Mu1307. 52

Figure 3.4. Fluctuation of aadB Amplification During and After Tobramycin Selection. 55

Figure 3.5. Resistant Subpopulation is Decreased in a recA Mutant. 57

Figure 3.6. Alignment of Illumina Data Suggests Amplified 21KB Region in Mu1307. 59

Figure 3.7. Tobramycin Heteroresistance Mediates Treatment Failure In Vivo. 60

Discussion. 61

Figure 3.8. AadB Mediates Treatment Failure in Heteroresistant Strain Mu1307. 62

Materials and Methods. 66

Supplemental Material 71

Supplementary Table 3.1. Carbapenem-Resistant Enterobacteriaceae (CRE) Isolate Patient Demographics. 71

Supplementary Table 3.2. Primers Used in This Study. 72

Supplementary Figure 3.1. Mu1307 Antibiogram. 73

Chapter 4: Discussion and Relevance. 74

Gene Amplification Mediated by Homologous Recombination. 75

Aminoglycoside Heteroresistance Dynamics. 76

Amplification-Independent Heteroresistance Mechanisms. 78

Figure 4.1. Summary of Work Presented in This Study. 81

Chapter 5: Appendix. 82

5A: Aminoglycoside heteroresistance in Acinetobacter baumannii AB5075. 82

Abstract 83

Introduction. 85

Results. 87

Discussion. 93

Materials and Methods. 96

Table 5A.1. The tobramycin resistant subpopulation exhibits increased cross-resistance to gentamicin but not other antimicrobials. 102

Table 5A.2. HetR-O2 and hetR-T1 exhibit gene amplifications that include aadB. 103

Table 5A.3. aadB copy number in the presence and absence of antibiotic selection. 104

Table 5A.4. Relative copy number of aadB in recA::Tc isolates with increased tobramycin resistance. 105

Figure 5A.1. AB5075 produces a subpopulation of cells with increased resistance to tobramycin. 106

Figure 5A.2. Characterization of tobramycin heteroresistance in AB5075. 108

Figure 5A.3. Increased expression of aadB in cells with increased tobramycin resistance. 109

Figure 5A.4. Amplified region in p1AB5075 that includes aadB. 110

Supplementary Table 5A.1. Oligonucleotides used in this study. 111

Supplemental Fig. 5A.S1. Complementation of recA leads to increased heteroresistance in the recA::Tc mutant 112

Supplemental Fig. 5A.S2. Characterization of tobramycin heteroresistance in a recA mutant 113

5B: mcr-1 Confers Cross-Resistance to the Cationic Host Antimicrobial Lysozyme. 114

Figure 5B.1. mcr-1 mediates resistance to lysozyme. 117

5C: Methods to Evaluate Colistin Heteroresistance in Acinetobacter baumannii 118

Abstract 119

Introduction. 119

Figure 5C.1. Sample population analysis profile (PAP) procedure using 96-well plate. 124

Figure 5C.2. Example graph of a typical population analysis profile with three strains of varying susceptibility. 126

Figure 5C.3. Example of Etest and disc diffusion assay results showing three different susceptibility profiles. 130

Acknowledgements. 136

References. 138

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