Department of Medical Biology, Faculty of Health Sciences Department of Microbiology, Vestfold Hospital Trust Non-beta-lactamase-mediated beta-lactam resistance in Haemophilus influenzae Mechanisms, epidemiology and susceptibility testing — Dagfinn Skaare A dissertation for the degree of Philosophiae Doctor – January 2016 Non-beta-lactamase-mediated beta-lactam resistance in Haemophilus influenzae Mechanisms, epidemiology and susceptibility testing DAGFINN SKAARE 2016 Department of Medical Biology, Faculty of Health Sciences University of Tromsø - The Arctic University of Norway Tromsø, Norway Department of Microbiology, Vestfold Hospital Trust Tønsberg, Norway 1 2 In the precious memories of my father, mother and brother 3 4 The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant. Alexander Fleming in his Nobel lecture, December 1945 [133] 5 6 TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................................... 11 SUMMARY ................................................................................................................. 15 ABBREVIATIONS ..................................................................................................... 19 PREFACE .................................................................................................................... 23 PART I. BACKGROUND 1 HISTORY............................................................................................................... 27 2 TAXONOMY, IDENTIFICATION AND CHARACTERIZATION .............. 31 2.1 Genus Haemophilus .............................................................................................. 31 2.2 The Haemophilus influenzae group ...................................................................... 33 2.2.1 Haemophilus influenzae .................................................................................. 34 2.2.2 Haemophilus aegyptius ................................................................................... 35 2.2.3 Haemophilus haemolyticus ............................................................................. 36 2.2.4 ‘Haemophilus quentini’ .................................................................................. 37 2.2.5 ‘Haemophilus intermedius’ ............................................................................ 38 2.3 Other Haemophilus species in humans ................................................................. 38 2.4 Factor X and factor V dependency ....................................................................... 39 2.5 Phenotypic characteristics .................................................................................... 40 2.6 Biochemical identification systems ...................................................................... 41 2.7 Biotyping .............................................................................................................. 42 2.8 Capsular serotyping .............................................................................................. 44 2.9 MALDI-TOF ........................................................................................................ 45 2.10 Molecular methods for species identification ..................................................... 46 3 GENETIC VARIATION AND EPIDEMIOLOGICAL TYPING ................... 49 3.1 To hit a moving target ........................................................................................... 49 3.2 Horizontal gene transfer ....................................................................................... 49 3.3 Competence and recombination ........................................................................... 50 3.4 Pulsed-field gel electrophoresis (PFGE) .............................................................. 53 3.5 Multilocus enzyme electrophoresis (MLEE) ........................................................ 54 3.6 Multilocus sequence typing (MLST) .................................................................... 54 3.7 Multilocus sequence analysis (MLSA) ................................................................. 57 3.8 Whole-genome sequencing (WGS) ...................................................................... 59 3.9 Consistency between typing methods ................................................................... 60 7 4 CLINICAL ASPECTS .......................................................................................... 63 4.1 Colonization and transmission .............................................................................. 63 4.2 Disease and epidemiology .................................................................................... 64 4.2.1 Encapsulated H. influenzae ............................................................................ 64 4.2.2 Nontypeable H. influenzae (NTHi) ................................................................. 65 4.3 Virulence and pathogenicity ................................................................................. 67 4.4 Biofilm .................................................................................................................. 69 4.5 NTHi vaccines ...................................................................................................... 70 4.6 Surveillance .......................................................................................................... 71 5 BETA-LACTAM ANTIBIOTICS AND THE CELL WALL ........................... 73 5.1 Definitions and characteristics .............................................................................. 73 5.2 Activity against H. influenzae ............................................................................... 74 5.3 Peptidoglycan biosynthesis ................................................................................... 77 5.4 Penicillin-binding proteins: Beta-lactam targets .................................................. 78 5.5 Penicillin-binding proteins in H. influenzae ......................................................... 81 5.5.1 Terminology .................................................................................................... 81 5.5.2 Essentiality ...................................................................................................... 84 5.5.3 Beta-lactam affinity ........................................................................................ 85 5.6 PBP3, cell division and the ftsI gene .................................................................... 88 5.7 Regulation of ftsI transcription and cell division .................................................. 90 6 BETA-LACTAM RESISTANCE ........................................................................ 93 6.1 Ampicillin therapy failure: early reports .............................................................. 93 6.2 Beta-lactamase-mediated resistance ..................................................................... 93 6.2.1 Emergence and spread ................................................................................... 93 6.2.2 TEM and ROB beta-lactamases ...................................................................... 94 6.2.3 Other beta-lactamases .................................................................................... 96 6.2.4 Beta-lactamase detection ................................................................................ 97 6.3 Penicillin-binding protein 3-mediated resistance ................................................. 98 6.3.1 Emergence and spread ................................................................................... 98 6.3.2 Clinical relevance ......................................................................................... 100 6.3.3 Characterization of the resistance mechanism ............................................. 100 6.3.4 Categorization and terminology ................................................................... 102 6.3.5 Resistance genotypes and phenotypic susceptibility profiles ....................... 105 6.3.6 Geographic distribution of rPBP3 genotypes .............................................. 108 6.3.7 Molecular epidemiology and clonal spread ................................................. 113 6.3.8 Evolution of rPBP3 ....................................................................................... 115 6.3.9 Molecular detection and genotyping ............................................................ 118 8 6.4 Other resistance mechanisms .............................................................................. 119 6.4.1 PBP-mediated resistance other than rPBP3 ................................................ 120 6.4.2 Impermeability .............................................................................................. 125 6.4.3 Increased efflux ............................................................................................. 127 6.5 Non-bla-mediated resistance and pathogenicity ................................................. 128 6.6 Beta-lactam resistance epidemiology ................................................................. 129 7 NON-BETA-LACTAM ANTIMICROBIAL AGENTS .................................. 131 7.1 Agents with activity against H. influenzae ......................................................... 131 7.2 Non-beta-lactam resistance in H. influenzae