Antimicrobial susceptibility and molecular epidemiology phd.· Antimicrobial susceptibility and molecular

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  • Antimicrobial susceptibility and molecular epidemiology

    of drug-resistant bacterial pathogens

    of healthcare-associated infections

    Doctoral thesis

    Katalin Glatz MD

    Semmelweis University

    Pathology School of Ph.D. Studies

    Supervisor: Dra Szab MD, D.Sc.

    Official referees:

    Ivelina Damjanova MD, Ph.D.

    Eszter Ostorhzi MD, Ph.D.

    Committee members of the Final Examination:

    Prof. Kroly Cseh MD, D.Sc. (chairman)

    Szabolcs Sipeki MD, Ph.D.

    Edit Urbn MD, Ph.D., Med. Habil.

    Budapest

    2012

  • 1

    I. Contents

    I. Contents .. 1

    List of tables 2

    List of figures ...... 3

    II. List of abbreviations .. 5

    III. Introduction ... 8

    IV. Objectives ..... 13

    IV.I. Drug-resistant Streptococcus pneumoniae ...... 13

    IV.II. Vancomycin-resistant Enterococcus spp. 13

    IV.III. MDR Acinetobacter baumannii ... 13

    IV.IV-VI. Enterobacter cloacae ...............14

    V. Materials and methods ... 16

    V.I. Clinical settings and bacterial strains .......16

    V.II. Biochemical identification and antimicrobial susceptibility testing ....19

    V.III. Phage typing ........ 21

    V.IV. Analytical isoelectric focusing ........ 21

    V.V. Organic solvent tolerance test . 22

    V.VI. Efflux-pump inhibitor test with Phe-Arg--naphtylamide (PAN) 22

    V.VII. Molecular typing methods ... 23

    V.VII.I. PFGE ... 23

    V.VII.II. MLVA and MLST .. 24

    V.VII.III. PCR based molecular epidemiological examinations ..... 24

    V.VII.IV. Plasmid profile analysis .. 26

    V.VIII. Specific PCR assays .... 26

    V.IX. Analysis of DNA gels ..................... 27

    V.X. Mating out assays ... 27

    V.XI. Statistical analyses ..... 28

    VI. Results . 30

    VI.I. Drug-resistant Streptococcus pneumoniae .......... 30

    VI.II. Vancomycin-resistant Enterococcus spp. .... 42

    VI.III. Multidrug-resistant Acinetobacter baumannii ..... 46

    VI.IV. Multidrug-resistant Enterobacter cloacae .......... 50

  • 2

    VI.IV.I. Emergence of SHV-2a producing Enterobacter cloacae in Hungary . 50

    VI.IV.II. Results of the national ESBL surveillance (2002-2004) and the

    examination of the outbreak strain of the first reported ESBL

    Enterobacter cloacae outbreak in Hungary ... 53

    VI.IV.III. Cyclohexane tolerance and Phe-Arg--naphtylamide susceptibility of

    MDR Enterobacter cloacae clinical isolates, predominance of one PFGE

    clone in Hungary, report of a nationwide survey 59

    VII. Discussion ....... 72

    VIII. Conclusions . 99

    IX. Summary ...... 100

    X. References ........ 102

    XI. List of publications ...... 125

    XII. Acknowledgements . 126

    List of tables

    VI.I. Drug-resistant Streptococcus pneumoniae

    Table I. Antimicrobial susceptibility of all the tested S. pneumoniae and

    PRSP strains .. 31

    Table II. a Antimicrobial activity (mg/L) of penicillin, cefotaxime and

    levofloxacin on respiratory tract PNSP isolates 34

    Table II. b Cefotaxime and levofloxacin susceptibility of PNSP strains

    isolated from the respiratory tract .. 35

    Table III. Penicillin, cefotaxime, carbapenem, and vancomycin MICs of

    S. pneumoniae strains with extremely high penicillin and/or cefotaxime

    MIC.................................................................................................... 37

    Table IV. Results of comparative testing of penicillin and cefotaxime

    MICs with E-test and agar dilution on 12 extremely resistant Hungarian

    S. pneumoniae isolates .. 38

    Table V. Penicillin, cefotaxime, and levofloxacin MICs (mg/L) of

    S. pneumoniae isolates derived from patients treated at the Department

    of Pulmonology in the study period . 39

  • 3

    VI.II. Vancomycin-resistant Enterococcus spp.

    Table VI. Antimicrobial susceptibility patterns (P1-P7) displayed by the

    VanB E. faecium outbreak strain ...... 44

    VI.III. Multidrug-resistant Acinetobacter baumannii

    Table VII. Results of PCR based typing, PFGE, and antimicrobial

    susceptibility testing ..... 49

    VI.IV. Multidrug-resistant Enterobacter cloacae

    Table VIII. Laboratory results by antimicrobial susceptibility testing and

    ERIC-PCR (n=142) .. 51

    Table IX. Clinical data and laboratory results for the confirmed ESBL

    isolates 54-56

    Table X. MIC values of the outbreak isolates, the transconjugants,

    and the recipient strain .. 58

    Table XI. Clinical data and results of laboratory tests performed on all

    the isolates ....... 60-65

    Table XII. a Ciprofloxacin, chloramphenicol, and tetracycline MIC

    values plotted against the cyclohexane (CH) tolerance .... 69

    Table XII. b Ciprofloxacin, chloramphenicol, and tetracycline MIC

    values plotted against the PAN susceptibility 70

    List of figures

    VI.I. Drug-resistant Streptococcus pneumoniae

    Figure I. Distribution of isolates by the site of infection ......... 30

    Figure II. Distribution of PNSP strains by their penicillin MICs ................... 32

    Figure III. Penicillin susceptibility of S. pneumoniae strains (n=327)

    distributed by age .. 33

    Figure IV. Protocol for the quantitative antimicrobial susceptibility testing

    of respiratory tract isolates of S. pneumoniae ... 33

    Figure V. Distribution of penicillin intermediate-susceptible and resistant

    strains of S. pneumoniae isolated from the respiratory tract according to

    their cefotaxime (a) and levofloxacin (b) MICs (n = 96) ..... 36

    Figure VI. a The line laid with the Statistica for Windows software

  • 4

    on the penicillin MICs vs cefotaxime MICs got by the Spearman

    rank correlation analysis ... 40

    Figure VI. b The line laid with the Statistica for Windows software

    on the penicillin MICs vs levofloxacin MICs got by the Spearman

    rank correlation analysis ... 41

    Figure VI. c The line laid with the Statistica for Windows software

    on the cefotaxime vs levofloxacin MICs got by the Spearman rank

    correlation analysis ... 41

    VI.II. Vancomycin-resistant Enterococcus spp.

    Figure VII. PFGE patterns of nine isolates derived from the NHI in 2004 . 43

    VI.III. Multidrug-resistant Acinetobacter baumannii

    Figure VIII. Patterns of the A. baumannii isolates with the ERIC-2 primer .. 47

    Figure IX. PFGE patterns of A. baumannii isolates 47

    Figure X. Class-1 integron patterns of the A. baumanni isolates . 48

    VI.IV. Multidrug-resistant Enterobacter cloacae

    Figure XI. Distribution of CREC strains (1998) by antibiogram and date

    of isolation 51

    Figure XII. a The three PFGE clones of the examined period .... 52

    Figure XII. b Different pulsopatterns of the predominant pulsotype

    (ERIC type I) .... 52

    Figure XIII. XbaI Pearsons hierarchical tree diagram for the 40 PFGE strains

    of type A ... 66

    Figure XIV. SpeI Dice hierarchical tree diagram for the selected isolates

    (n=10) and the Lambda concatemers ... 67

  • 5

    II. List of abbreviations

    AFLP amplified fragment length polymorphism

    AMR antimicrobial resistance

    AP-PCR arbitrarily-primed PCR

    BAZ Borsod-Abaj-Zempln

    Bp Budapest

    BSI bloodstream infection

    CC clonal complex

    CDC Centers for Disease Control

    CFU colony forming unit

    CH county hospital

    CNS Coagulase-negative staphylococci

    CNSI central nervous system infection

    CLSI Clinical Laboratory Standards Institute

    CML clinical microbiology laboratory

    CREC cephalosporine-resistant E. cloacae

    CVC central venous catheter

    EARSS European Antimicrobial Resistance Surveillance System

    Env environmental

    ERIC Enterobacterial repetitive intergenic consensus sequences

    ESBL extended-spectrum -lactamase

    EU European Union

    FQ fluoroquinolone

    HAI healthcare-associated infections

    HLAR high level aminoglycoside resistance

    ICU intensive care unit

    IEF isoelectric focusing

    MDR multidrug-resistant, multidrug resistance

  • 6

    List of abbreviations

    MIC minimal inhibitory concentration

    MH municipal hospital

    MLST multilocus sequence typing

    MLVA multiple locus variable-number tandem repeat analysis

    MMA Military Academy, Belgrad

    MRSA methicillin-resistant S. aureus

    NBS national bacteriological surveillance

    NCCLS National Center for Clinical Laboratory Standards

    NCE National Center for Epidemiology

    NHI National Health Institute

    NPHMOS National Public Health and Medical Officers System

    OEK Orszgos Epidemiolgiai Kzpont

    og obstetrics-gynecology

    PAN Phenylalanine-Arginin--naphtylamide

    PCV pneumococcus conjugate vaccine

    PFGE pulsed field gel electrophoresis

    PICU perinatal intensive care unit

    PNSP penicillin-nonsusceptible S. pneumoniae

    PREA plasmid restriction analysis

    PRSP penicillin-resistant S. pneumoniae

    PYR pyrrolidonyl-beta-naphtylamide

    QRDR quinolone-resistance determining region

    RAPD randomly amplified polymorphic DNA

    RFLP restric