DISSERTATION
Identification and characterization of novel carbapenemases
Dissertation to obtain the degree Doctor Rerum Naturalium (Dr. rer. nat.) at the Faculty of Biology and Biotechnology International Graduate School of Biosciences Ruhr-University Bochum Department of Medical Microbiology
submitted by Niels Ernst Pfennigwerth from Essen
Advisor: Prof. Dr. Sören G. Gatermann Second advisor: Prof. Dr. Franz Narberhaus
Bochum, April 2015 DISSERTATION
Identifizierung und Charakterisierung neuer Carbapenemasen
Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat) an der Fakultät für Biologie und Biotechnologie Internationale Graduiertenschule Biowissenschaften Ruhr-Universität Bochum Abteilung für Medizinische Mikrobiologie
eingereicht von Niels Ernst Pfennigwerth Essen
Referent: Prof. Dr. Sören G. Gatermann Korreferent: Prof. Dr. Franz Narberhaus
Bochum, April 2015
Danksagung
Viele haben zu einem erfolgreichen Gelingen dieser Dissertation beigetragen. Einigen möchte ich besonders danken.
Meinem Doktorvater Herrn Prof. Dr. Sören G. Gatermann danke ich sehr für seine fortwährende Unterstützung, sein großes Vertrauen in meine Arbeit und die Möglichkeit, in diesem interessanten Fachbereich zu promovieren.
Herrn Prof. Dr. Franz Narberhaus danke ich sehr für die freundliche Übernahme des Korreferats.
Herrn Dr. Alexander Stang und Herrn Prof. Klaus Überla danke ich für die Möglichkeit, das in dieser Arbeit gefundene Plasmid in der Abteilung für Virologie zu sequenzieren.
Allen Mitarbeitern der Abteilung für medizinische Mikrobiologie danke ich für das tolle, nette und freundschaftliche Arbeitsklima und für eine Hilfsbereitschaft, die nie zu enden scheint. Besonders danke ich hierbei Frau Anja Kaminski für die Hilfe bei den isoelektrischen Fokussierungen, Frau Anke Albrecht für ihre unverzichtbare Unterstützung bei den Lokalisationsstudien und Frau Susanne Friedrich für ein immer offenes Ohr bei experimentellen Problemen.
Danke auch an meine Masterstudent(in)en Lisei Meining, Alexander Hoffmann und Felix Lange und meine S-Moduler für ihr Mitwirken an Teilen dieser Arbeit.
Ein besonders großer Dank geht an meine KoMaNePf-Mitinsassen Dr. Sandra Neumann, Dr. Lennart Marlinghaus und Dr. Miriam Korte-Berwanger, ohne euch wären die letzten vier Jahre um mindestens 90% unlustiger gewesen. Auch für viele fachliche Diskussionen - vielen Dank!
(Fast) last, but not least: Ein riesiggroßer Dank geht an Herrn Dr. Martin Kaase für seine zu jeder Zeit freundschaftliche Unterstützung, die zahllosen fruchtbaren fachlichen Diskussionen, das kritische Korrekturlesen von Postern, Manuskripten und dieser Arbeit und als wandelndes Lexikon für alle Fragen bezüglich der medizinischen Mikrobiologie. Vielen Dank!
Ein Dank, der so groß ist, dass ich ihn nicht in Worten auszudrücken vermag, gebührt zu guter Letzt meinen Eltern, meiner Schwester und meiner Frau Freya, die mich zu jeder Zeit bedingungslos unterstützt, ermutigt und aufgebaut haben. Vielen, vielen Dank! Contents I
Contents
Contents ...... I
List of Figures ...... IV
List of Tables ...... V
Abbreviations ...... VI
1 Introduction ...... 1
1.1 -lactam antibiotics ...... 1
1.2 β -lactam antibiotics: The bacterial cell wall synthesis ...... 5
1.3 TargetMechanisms structures of antibiotic of β resistance ...... 8
1.4 -lactamases ...... 10
1.4.1β -lactamases ...... 12
1.4.2 Class A β-lactamases ...... 13
1.4.3 Class BC β-lactamases ...... 14
1.4.4 β-lactamases ...... 14
1.5 CarbapenemasesClass D β and their distribution ...... 15
1.6 -lactamase genes ...... 16
1.7 PseudomonasMobility of β aeruginosa ...... 19
1.8 Citrobacter freundii ...... 19
1.9 Objectives of this work ...... 20
2 Material and Methods ...... 22
2.1 Material ...... 22
2.1.1 Instruments ...... 22
2.1.2 Disposable material ...... 23
2.1.3 Chemicals ...... 24
2.1.4 Antibiotics ...... 25
2.1.5 Wafers containing antibiotics ...... 26
2.1.6 Antibiotic gradient test strips ...... 26 Contents II
2.1.7 Kits und standards ...... 26
2.1.8 Enzymes ...... 27
2.1.9 Antibodies ...... 27
2.2 Microbial strains, plasmids and oligonuclotides ...... 28
2.2.1 Microbial strains ...... 28
2.2.2 Plasmids ...... 28
2.2.3 Oligonucleotides ...... 29
2.3 Methods ...... 37
2.3.1 Microbiological methods ...... 37
2.3.2 Phenotypic methods for antibiotic resistance analysis ...... 39
2.3.3 Molecular biology methods ...... 40
2.3.4 Biochemical methods ...... 46
2.3.5 In silico methods ...... 50
3 Results ...... 51
3.1 The search for novel carbapenemases ...... 51
3.1.1 Identification of IMP-31 in Pseudomonas aeruginosa NRZ-00156 ...... 51
3.1.2 Identification of OXA-233 in Citrobacter freundii NRZ-02127 ...... 55
3.1.3 Identification of KHM-2 in Pseudomonas aeruginosa NRZ-03096 ...... 58
3.2 Analysis of the genetic environment of blaIMP-31, blaOXA-233 and blaKHM-2 ...... 61
3.2.1 Genetic environment of blaIMP-31 ...... 61
3.2.2 Genetic environment of blaOXA-233 ...... 62
3.2.3 Genetic environment of blaKHM-2 ...... 63
3.3 Localization of blaIMP-31, blaOXA-233 and blaKHM-2 ...... 64
3.3.1 Localization of blaIMP-31 ...... 64
3.3.2 Localization of blaOXA-233 ...... 65
3.3.3 Localization of blaKHM-2 ...... 66
3.4 Impact of IMP-31, OXA-233 and KHM- -lactam resistance ...... 67
3.4.1 Impact of IMP- -lactam resistance2 on β ...... 68
3.4.2 Impact of OXA-31 on β -lactam resistance ...... 69
233 on β Contents III
3.4.3 Impact of KHM- -lactam resistance ...... 72
3.4.4 Comparison of IMP2 on-31, β OXA-233 and KHM-2 ...... 73
3.5 Purification of IMP-31, OXA-233 and KHM-2 ...... 74
3.6 Determination of kinetic parameters ...... 77
3.6.1 Determination of kinetic parameters for IMP-31 ...... 77
3.6.2 Determination of kinetic parameters for OXA-233 ...... 79
3.6.3 Determination of kinetic parameters for KHM-2 ...... 81
3.6.4 Comparison of the hydrolytic efficiencies of IMP-31, OXA-233 and KHM-2 ...... 82
3.7 Determination of the isoelectric point of IMP-31, OXA-233 and KHM-2 ...... 83
3.8 Sequencing and characterization of the blaOXA-233 carrying plasmid pMB3018 ...... 84
4 Discussion ...... 89
4.1 Identification of IMP-31 ...... 89
4.2 Identification of OXA-233 ...... 94
4.3 Identification of KHM-2 ...... 95
4.4 Catalytic characteristics of IMP-31, OXA-233 and KHM-2 ...... 98
4.4.1 Characteristics of IMP-31 ...... 98
4.4.2 Characteristics of OXA-233 ...... 101
4.4.3 Characteristics of KHM-2 ...... 106
4.5 Characterization of the blaOXA-233-carrying plasmid pMB3018 ...... 108
4.6 Comparison of IMP-31, KHM-2 and OXA-233 and concluding remarks ...... 109
5 Summary...... 111
6 Zusammenfassung ...... 113
7 Bibliography ...... 115
8 Appendix...... 132
Publications ...... 137
Curriculum vitae ...... 139
List of Figures IV
List of Figures
-lactam antibiotics...... 3
Figure 1.21.1 Chemical structures of imipenem,the backbone meropenem, of β ertapenem and doripenem...... 4 Figure 1.3 Chemical structure of peptidoglycan from E. coli...... 6 -lactamase against carbapenems...... 12
Figure 1.51.4 SchematicAction of a organizationserine β of transporter insertion sequences and transposons. (A) ...... 17 Figure 1.6 Schematic structure of a class 1 integron...... 19 Figure 3.1 Modified Hodge Test and EDTA-CDT of P. aeruginosa NRZ-00156...... 51 Figure 3.2 Amino acid sequence alignment of IMP-31, IMP-35 and IMP-1...... 53 Figure 3.3 Phylogenetic analysis of IMP-31...... 54 Figure 3.4 Modified Hodge Test of C. freundii NRZ-02127...... 55 Figure 3.5 Amino acid sequence alignment of OXA-233, OXA-17 and OXA-10...... 57 Figure 3.6 Modified Hodge Test and EDTA-CDT of P. aeruginosa NRZ-03096...... 58 Figure 3.7 Amino acid sequence alignment of KHM-2 and KHM-1...... 60
Figure 3.8 Genetic environment of blaIMP-31 in P. aeruginosa NRZ-00156...... 61
Figure 3.9 Genetic environment of blaOXA-233 in C. freundii NRZ-02127...... 62
Figure 3.10 Genetic environment of blaKHM-2 in P. aeruginosa NRZ-03096...... 63
Figure 3.11 Localization of blaIMP-31...... 65
Figure 3.12 Localization of blaOXA-233...... 66
Figure 3.13 Localization of blaKHM-2...... 67 Figure 3.14 Ion exchange (A) and gel filtration (B) chromatograms of the KHM-2 FPLC...... 75 Figure 3.15 SDS-PAGE analysis of enzyme preparations of IMP-31, IMP-1, OXA-233, OXA-10, KHM-2 and KHM-1...... 76 Figure 3.16 Hydrolysis assay of IMP-31 for imipenem and Michaelis-Menten plot...... 78 Figure 3.17 CO2-dependent imipenem hydrolysis of OXA-233...... 80 Figure 3.18 Isoelectric focussing of OXA-233, OXA-10, IMP-31, IMP-1, KHM-2 and KHM-1...... 84 Figure 3.19 Circular map of pMB3018...... 85 Figure 3.20 Comparison of pMB3018, pJIE137, p271A, pECS01 and pTR3...... 87
Figure 4.1 Comparison of the genetic environment of blaIMP-31 and blaIMP-35...... 92 Figure 4.2 Crystal structure and homology model of the active site of IMP-1 (A) and IMP-31 (B)...... 100 Figure 4.3 Crystal structure and homology model of the active sites of OXA-10 (A) and OXA-233 (B)...... 104 Figure 4.4 Chemical structures of ceftazidime, aztreonam and penicillin G...... 105 Figure 4.5 Homology models of KHM-1 (A) and KHM-2 (B)...... 107 List of Tables V
List of Tables
-lactamases according to Bush & Jacoby (2010) and Ambler
Table(1980). 1.1 ...... Classification ...... schemes for β ...... 11 Table 2.1. Microbial strains used in this study...... 28 Table 2.2. Plasmids used in this study...... 28 Table 2.3: Oligonucleotides used in this study...... 29 -lactam MICs of P. aeruginosa NRZ-00156...... 52
Table 3.23.1 MLSβ typing of P. aeruginosa NRZ-00156...... 55 -lactam MICs of C. freundii NRZ-02127...... 56
Table 3.3 β-lactam MICs of P. aeruginosa NRZ-03096...... 59 Table 3.53.4 MLSβ typing of P. aeruginosa NRZ-03096...... 60 -lactam MICs of E. coli TOP10 transformed with the pBK-CMV vector and
TableIMP-31/IMP 3.6 β-1 expressing E. coli TOP10...... 68 -lactam MICs of E. coli TOP10 transformed with the pBK-CMV vector and
TableOXA-233/OXA 3.7 β -10 expressing E. coli TOP10...... 70 -lactam MICs of the E. coli C600 OXA-233 pMB3018-transconjugant and E. coli C600.
Table...... 3.8 β ...... 71 -lactam MICs of E. coli TOP10 transformed with the pBK-CMV vector and
TableKHM- 2/KHM 3.9 β-1 expressing E. coli TOP10...... 72 Table 3.10 Relative MIC increases of E. coli TOP10 producing IMP-31, OXA-233 and KHM-2...... 73 Table 3.11 Kinetic parameters of IMP-31...... 79 Table 3.12 Kinetic parameters of OXA-233...... 80 Table 3.13 Kinetic parameters of KHM-2...... 81 Table 3.14 Comparison of the hydrolytic efficiencies of IMP-31, OXA-233 and KHM-2...... 83
Abbreviations VI
Abbreviations
All abbreviations that are not listed here are either part of the International System of Units (Système international d’unités, SI) or abbreviations of chemicals that are mentioned in the Materials and Methods section (Chapter 2).
aa Amino acid A. dest Aqua destilata (lat.), distilled water AMP Ampicillin AmpR Ampicillin resistance AP Alkaline phosphatase BLAST Basic Local Alignment Search Tool bp Base pairs BSA Bovine serum albumin CHDL Carbapenem- -lactamase
CDT Combined-diskhydrolyzing test class D β DNA Deoxyribonucleid acid ECDC European Centre for disease prevention and control ESBL Extended- -lactamase
ETP Ertapenemspectrum β EUCAST European Committee on Antimicrobial Susceptiblity Testing FOX Cefoxitin FPLC Fast protein liquid chromatography GF Gel filtration HAI Healthcare-associated infections IEF Isoelectric focussing IEX Ion exchange IMP Imipenem IR Inverted repeats kb kilo base pairs KmR Kanamycin resistance mAU milli absorbance units MBL metallo- -lactamase
Mbp Mega baseβ pairs Abbreviations VII
MCS Multiple cloning site MDR Multidrug-resistant MEM Meropenem MIC Minimal inhibitory concentration NCBI National Centre for Biotechnology Information NRZ National Reference Laboratory for multidrug-resistant Gram-negative bacteria (“Nationales Referenzzentrum für Gram-negative Krankenhauserreger”) OD Optical density ORF Open reading frame PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction PFGE Pulsed-field gel electrophoresis pI Isoelectric point RifR Rifampicin resistance (r)RNA (ribosomal) Ribonucleic acid TBE Tris-boric acid-EDTA buffer v/v volume per volume w/v weight per volume
Introduction 1
1 Introduction
Antibiotic resistance in clinically relevant bacteria is a major challenge to healthcare systems worldwide. Especially the ongoing spread and diversification of resistance mechanisms in Gram- negative pathogens is a worrying development. Gram-negative pathogens, such as Escherichia coli, Klebsiella pneumoniae, other members of the Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii can cause severe infections and are a major threat to critically ill hospitalized patients (Gaynes & Edwards, 2005). Studies of the European Centre for Disease Prevention and Control (ECDC) estimated that 1.9 to 5.2 million patients per year in Europe are infected with bacterial pathogens in context of a medical treatment and that 75 % of these healthcare-associated infections (HAI) result from hospitalization (Suetens et al., 2013). In an ECDC surveillance study with data from over 250,000 patients affected by HAI in 2011 and 2012, infections with E. coli were the most prevalent with 15.9 %, followed by Staphylococcus aureus infections with 12.3 % (Suetens et al., 2013). While the main focus of antimicrobial treatment of the last decades was set on S. aureus infections, especially with the methicillin-resistant S. aureus (MRSA), the most threatening development nowadays is the increasing number of Gram-negative pathogens that are resistant to antibiotics (ECDC, 2013; Suetens et al., 2013). Many Gram-negative species are intrinsically resistant to single antibiotics, but in the last decades, these pathogens have acquired numerous resistance genes, becoming multidrug-resistant (MDR) or pan-resistant and limiting the treatment options in many cases dramatically (Falagas & Bliziotis, 2007). In this context, antibiotic resistance has been listed as one of the greatest threats to human health in the most recent World Economic Forum Global Risks Reports (World Economic Forum, 2013 & 2014). As there is a lack in development of novel antibiotics against Gram-negative pathogens due to economical and organizational reasons (Appelbaum, 2012) and as only few novel antibacterial drugs are expected to be clinically available in the next years, the situation is predicted to escalate further (Boucher et al., 2013). In this context, the identification and characterization of resistance mechanisms in Gram-negative bacteria and the correct treatment of patients infected with these bacteria in combination with strict hygiene management is the major challenge to antimicrobial treatment and infection control precautions for the next years.
1.1 β-lactam antibiotics -lactam antibiotics are the most important class of antibiotics and were first discovered in 1929 byβ Sir Alexander Fleming, as he observed the inhibitory effect of a Penicillium notatum mycelium that contaminated a Staphylococcus colony on an agar plate. Although Fleming was not the first to observe the antibiosis between fungal and bacteria, he was the first to study one of the Introduction 2 substances that inhibit bacterial growth and named it penicillin (Kong et al., 2010). In 1940, penicillin was purified at higher levels and was sucessfully used to treat patients with S. aureus infections. Penicillin became finally available in the open market in 1946 (Kong et al., 2010). Several derivatives of penicillin were found or developed in the following decades, constituting -lactams: the penicillins, the cephalosporins, the carbapenems and the monobactamsfour groups of(Kong β et al., 2010; Papp-Wallace et al., 2011).
Penicillins -lactams in clinical use and were widely used in the beginning of
Thethe antibioticpenicillins era. were Th thee structure first β of the molecules is based upon the four-membered -lactam ring and an annulated five-membered thiazolidine ring with varying side chains (Figure β1.1). The thiazolidine ring exhibits sulfur at position C-1. Penicillins are classified into several groups based upon their origin. The natural penicillins benzylpenicillin (Penicillin G) and phenoxymethylpenicillin (Penicillin V) were isolated from different variants of Penicillium chrysogenum and are higly active against sensitive strains of Gram-positive cocci, therefore sparing most current strains of S. aureus (Mascaretti, 2003). Methicillin on the other hand -lactamase-resistant penicillin and was widely used in therapy againstis S.an aureus antistaphylococcal infections but β is no longer available nowadays. Other members of this group are the isoxazolyl-penicillins oxacillin, cloxacillin and dicloxacillin. The aminopenicillins include ampicillin, bacampicillin and amoxicillin. They have a broader spectrum, including several Gram- negatives like E. coli or Proteus mirabilis, as they are more capable of penetrating the outer membrane of these bacteria (Mascaretti, 2003). The last group are the antipseudomonal pencillins, which are semisynthetic derivates of penicillanic acid. They are categorized into two subgroups: the carboxypenicillins, including carbenicillin and ticarcillin and the ureidopenicillins, which include piperacillin and mezlocillin. Notably, piperacillin shows high activity against P. aeruginosa and Enterobacteriaceae, making it an important treatment option for infections with these species (Mascaretti, 2003).
Cephalosporins The first cephalosporin, cephalosporin C, was isolated in 1953 from Cephalosporium acremonium and the structure was determined in 1961 (Abraham & Newton, 1961). Cephalosporins consist -lactam ring, an annulated six-membered dihydrothiazine ring and two varying side ofchains the β(Figure 1.1). They are categorized into four to five generations based upon their -lactamases and membrane characteristicspenetrability (Mascaretti, regarding 2003 antimicrobial). The first activity, generation resistance includes to cephalotin, β cefazolin and others
Introduction 3
Figure 1.1 Chemical structures of the backbone of β-lactam antibiotics. -lactam antibiotics share the four- -lactam ring. Penicillins and cephalosporins possess a sulfur in the annulated thiazolidine ring while carbapenems exhibit a carbon at this position. In cephalosporins, the thiazolidineAll β ring is six-membered, while it is fivemembered-membered β in penicillins and carbapenems. that show high antibacterial activity against Gram-positive cocci, but are less effective against E. coli, P. mirabilis and Klebsiella pneumoniae. The second generation is subgrouped and includes the true cephalosporins, the cephamycins and the carbacephems. The cephalosporins of this group exhibit higher activity against Haemophilus influenzae, Neisseria meningitidis, staphylococci and streptococci than first-generation cephalosporins. An example for this group is cefuroxime. Cephamycins on the other hand show increased antibacterial action against Gram- negative bacteria and Bacteroides spp. and possess a –OCH3 group as a third side chain, -lactamases and their antibacterial activity. They are less increasingeffecive against their staphylococcistability to certain and stre β ptococci (Mascaretti, 2003). Examples for clinically used cephamycins are cefoxitin and cefotetan. Loracarbef is the only carbacephem and is not a true cephalosporin but closely related. The third-generation cephalosporins, or oxyimino- cephalosporins, exhibit significantly higher activity against Gram-negative bacteria than the first and second generations. -lactamases and have a broader spectrum, including E. coli, KlebsiellaThey are spp., more P. stable mirabilis to β, Citrobacter spp., Serratia marcescens, Streptococcus pneumoniae, Streptococcus pyogenes and others (Mascaretti, 2003). Clinically important members of this generation are cefotaxime, ceftriaxone and ceftazidime. The fourth generation of cephalosporins is characterized by higher antimicrobial activity against some Enterobacteriaceae, with cefepime and cefpirome being the only members of this generation (Mascaretti, 2003). Two novel cephalosporins with activity against MRSA are ceftobiprole and ceftaroline, which are classified as the fifth generation of cephalosporins (Bush et al., 2007; Saravolatz et al., 2011).
Carbapenems The first carbapenem, thienamycin, was discovered in 1976 in Streptomyces cattleya and served as the model compound for all carbapenems. In contrast to many penicillins and cephalosporins, the antimicrobial activity was shown for a broad range of bacteria, including even Gram- -lactams, like P. aeruginosa (Tally et negative organisms that are intrinsically resistant to many β Introduction 4 al., 1978; Weaver et al., 1979; Fainstein et al., 1982). In contrast to penicillins and cephalosporins, the carbapenems exhibit a carbon for sulfur substitution at position C-1 of the five-membered annulated ring (Figure 1.1). This carbon atom is responsible for the increased -lactamases and the broad- -lactams (Papp-Wallace et stabilityal., 2011 against). As thienamycin β was unstable in spectrumaqueous solutions,of this class the of searchβ for derivatives was intensified, leading to the development of imipenem. Imipenem became clinically available in -lactamases (Hashizume et al.,
19851984; andKong demonstrated et al., 2010). highImipenem target isaffinity the N -andformimidoyl stability againstderivative β of thienamycin (Figure 1.2) and is active against many Gram-positive and Gram-negative species. It has an increased inhibitory effect on most members of the Enterobacteriaceae and can be used to treat P. aeruginosa infections when combined with an aminoglycoside (Mascaretti, 2003). As imipenem is metabolized by the human renal dehydropeptidase-1 (DHP-1), it is combined with an inhibitor of this enzyme, cilastatin, in therapeutic use (Kropp et al., 1982; Norrby et al., 1983). Today, three other carbapenems besides imipenem are in clinical use: meropenem, ertapenem and doripenem. Meropenem possesses a 1- -methyl group on position C-1 of the carbapenem backbone (Figure 1.2) and is active against βa broad range of Gram-positive and Gram-negative pathogens with slightly elevated activity against Gram-negatives compared to imipenem. It is significantly more stable against degradation by DHP-1 (Mascaretti, 2003) due to the 1- -methyl group. Ertapenem also possesses a 1- -methyl group on position C-1 (Figure 1.2) and βhas high activity against many Gram-positiveβ and Gram-negative bacteria, but is weak against
Figure 1.2 Chemical structures of imipenem, meropenem, ertapenem and doripenem. The structure is based -lactam ring and an annulated five-membered thiazolidine ring. In contrast to imipenem, meropenem, ertapenem and doripenem possess a methyl group at position C-1 of the thiazolidine ring, confering stability against uponthe human the β renal dehydropeptidase DHP-1. Introduction 5
Acinetobacter spp. and Pseudomonas aeruginosa (Zhanel et al., 2005; Burkhardt et al., 2007). Doripenem on the other hand shows excellent activity against P. aeruginosa but also reduced activity against Acinetobacter spp. (Paterson & Depestel, 2009). The structure of doripenem is very similar to meropenem, with the dimethylcarbamoyl side chain of meropenem replaced with a sulfamoylaminomethyl group in doripenem (Figure 1.2). Carbapenems are considered as antibiotics of last resort and should exclusively be used for therapy of critically ill patients infected with multidrug-resistant bacteria that are still susceptible to carbapenems (Papp-Wallace et al., 2011).
Monobactams Monobactams are characterized by their molecular structure, which exhibits a four-membered -lactam ring without any annulated secondary ring structure in contrast to the bicyclic penicillins,β cephalosporins and carbapenems (Singh, 2004). The only clinically available member of this group is aztreonam, a totally synthetic antibiotic. It has specific activity against a wide -lactamase-producing Gram-negative bacteria, including P. aeruginosa (Mascaretti, range2003) . ofFurthermore, β a -lactamases and has a high and exclusive affinity for theztreonam PBP3 transpeptidaseshows increased of stabilityGram-negative to β bacteria, also known as FtsI (Mascaretti, 2003; Kong et al., 2010).
1.2 Target structures of β-lactam antibiotics: The bacterial cell wall synthesis -lactam antibiotics is the inhibition of cell wall synthesis in Gram-positive andThe modeGram -ofnegative action ofbacteria. β The cell wall of bacteria is located outside of the cytoplasmic membrane of almost all bacteria and protects the cell integrity by withstanding the turgor (Vollmer et al., 2008). The cell shape is also influenced by the cell wall and it is important for the anchoring of other components of the cell envelope, for example transmembrane proteins (Dramsi et al., 2008) or teichonic acids (Neuhaus & Baddiley, 2003). While cell walls are found in nearly every bacterial species that is clinically relevant, they are absent in Mycoplasmas, Planctomyces, Rickettsia spp. and Chlamydiae (Vollmer et al., 2008). The cell wall is formed by layers of the polymeric molecule peptidoglycan, which is illustrated in Figure 1.3. Peptidoglycan is formed by chains of repeating units of the disaccharide N-acetylglucosamine-N-acetylmuramic acid (GlcNAC-MurNAc) that are cross-linked by short polypeptides, while the saccharides are linked by - 4 bonds (Vollmer et al., 2008; Silhavy et al., 2010). The cross-linking peptide stem is most oftenβ 1→ formed by L-Ala- -D-Glu-meso-A2pm-D-Ala-D-Ala, where diaminopimelic acid (A2pm) can be replaced by L-Lys. Theγ terminal D-Ala is present only in the nascent molecule and is lost in the mature form (Vollmer et al., 2008). The cross-linking occurs between the carboxyl group of D-Ala and the amino group of the diaminopimelic acid or lysine and the peptide stems
Introduction 6
Figure 1.3 Chemical structure of peptidoglycan from E. coli. The N-acetylglucosamine-N-acetylmuramic acid layers are cross-linked by a L-Ala- -D-Glu-meso-A2pm-D-Ala polypeptide. The single components of the peptide are colored. Figure reproduced and modified from Mengin-Lecreulx & Lemaitre (2005). γ are substituted to the D-lactoyl group of each MurNAc residue (Figure 1.3). In the Gram-positive cell wall the multilayer is typically between 15 and 30 nm thick and additionally contains teichoic or teichuronic acids. In Gram-negative bacteria the cell was is located in the periplasmic space between the cytoplasmic membrane and the outer membrane and consists of thinner layers with diameters ranging from 2 to 6 nm depending on the species (Vollmer et al., 2008). As it has been shown that a single peptidoglycan layer has a diameter of approx. 2 to 2.5 nm (Labischinski et al., 1991), the cell wall of Gram-positive bacteria consists of up to 15 layers, while the Gram-negative cell wall exhibits only up to three layers (Matias et al., 2003). The biosynthesis of peptidoglycan is very similar in Gram-positive and Gram-negative bacteria. The first steps take place in the cytoplasm, where the synthesis of the GlcNAc and MurNAc precursors UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramic acid (UDP- MurNAc) is catalyzed by the enzymes MurA and MurB (Mascaretti, 2003). UDP-GlcNAc and UDP- MurNAc are then translocated to the cytoplasmic membrane and fused to each other to build the linear chain. Following, the peptide stem residues L-Ala, D-Glu, meso-A2pm and D-Ala are linked to the chain. These steps are catalyzed by the enzymes MurC, MurE and MurF (Mascaretti, 2003; Vollmer et al., 2008). In Gram-positive bacteria, the final cross-linking step takes place in in the extracellular space, while in Gram-negative bacteria it is catalyzed in the periplasmic space by one or more D-alanyl-D-alanine transpeptidases and a D-alanine carboxypeptidase that link the Introduction 7 lineal peptidoglycan chain units. The D-alanyl-D-alanine transpeptidases and the D-alanine carboxypeptidase are also known as penicillin-binding-proteins (PBPs), as they are the primary -lactam antibiotics (Mascaretti, 2003).
Severaltarget of PBPs β have been described -lactam affinity and vary from species to species. In E. coli, six PBPs,, which PBP1 significantly to PBP6, were differ identified in β (Spratt & Pardee, 1975) and numbered descending according to their molecular weight (Mascaretti, 2003). Similar numbers were found in P. aeruginosa, Enterobacter cloacae, Salmonella typhimurium and S. marcescens (Georgopapadakou & Liu, 1980; Kong et al., 2010). Gram-positive cocci on the other hand possess only four PBPs, while some Bacillus species express up to eight (Suginaka et al., 1972). Especially the low molecular mass PBPs, PBP5, PBP6 and PBP7 were only found in bacilli (Georgopapadakou & Liu, 1980). PBP1 of E. coli is subdivided into three components, PBP1a, PBP1b and PBP1c (Spratt & Jobanputra, 1977; Schiffer & Holtje, 1999). PBP1a and PBP1b function as transglycosylases and transpeptidases, while PBP1c is only a transglycosylase and the exact function of PCP1c is not known (Sauvage et al., 2008). PBP1-like enzymes catalyze the peptidoglycan synthesis at the growing zones of the cell wall sides and are effectively inhibited by pencillin G, most cephalosporins, imipenem and doripenem (Mascaretti, 2003; Breilh et al., 2013). PBP2 and PBP3 are transpeptidases. While PBP2 catalyzes the initiation of peptidoglycan insertion at growth sites, PBP3 is needed for formation of the cross-wall at cell division (Spratt, 1975; Mascaretti, 2003; den Blaauwen et al., 2008). PBP2 is one of the main target structures of all carbapenems, whereas PBP3 strongly binds many cephalosporins, piperacillin, meropenem, doripenem and aztreonam (Mascaretti, 2003; Breilh et al., 2013). The lower molecular mass PBPs of E. coli play a role in cell separation, peptidoglycan maturation or recycling (Sauvage et al., 2008). PBP4 (divided into PBP4a and PBP4b) and PBP7 function as endopeptidases that cleave cross-bridges between two glycan chains. PBP5 is the major carboxypeptidase that cleaves the terminal D-Ala-D-Ala bond. This cleavage prevents the transpeptidation of the stem peptide (Sauvage et al., 2008). The role of PBP6a and PBP6b is not completely understood, but both enzymes are carboxypeptidases like PBP5 and are assumed to be involved in the control of peptidoglycan extent and/or peptidoglycan recycling (Sauvage et al., 2008). While PBP4a and PBP4b have high affinity for penicillin G, ampicillin and imipenem, PBP5 is a major target structure of cefoxitin and imipenem. PBP7 has high affinity for all carbapenems (Mascaretti, 2003; Breilh et al., 2013).
Inhibition of PBPs by β-lactam antibiotics The bacterial cell wall is subject to permanent maintenance, controlled degradation and resynthesis. An inhibition of the essential enzymes involved in this process inevitably leads to instability of the wall, resulting in lysis and cell death (Mascaretti, 2003). The inactivation of -lactams and therewith the inhibition of cell wall synthesis is based upon the covalent
PBPs by β Introduction 8 binding and the formation of a stable acyl-ester between the PBP and the antibiotic (Zapun et al., 2008). -lactams mimic the D-Ala-D-Ala dipeptide necessary for peptidoglycan crosslinking and are boundβ by the PBPs. The active site serine of the PBP attacks the carbonyl group - lactam ring which leads to the opening of the ring and covalent binding to the enzyme.of As the this β complex is hydrolyzed with extremely low efficiency, it is equivalent to an inactivation of the enzyme (Zapun et al., 2008). From crystal structure analysis, several PBP- -lactam binding characteristics were analyzed, showing similarities to the PBP4a- -aminopimelylβ - -D-alanyl acyl anzyme and therewith the binding of PBPs to cell wall componentsα (Sauvage et al.,ε 2008). Crystal structures showed that the active site serine of PBPs is covalently linked to the antibiotic and the -lactam side chain is inserted between the second motif and the backbone amide group of the β -associated carboxylate binds to one ofor theboth β3 hydroxyl sheet of groupsthe PBP. of In the addition, PBPs KTGT the thiazolidine motif. As a thirdring characteristic, the carbonyl oxygen -lactam lies in the oxyanion hole of the PBP (Sauvage et al., 2008). As the PBP4 enzymes, ofPBP5 the, βthe PBP6 enzymes and PBP7 are not essential for growth in E. coli (Denome et al., 1999), -lactam antibiotics is based upon the inhibition of the PBP1 enzymes, thePBP2 bacteriolytic and PBP3 ( Mascaretti,effect of β 2003; Sauvage et al., 2008).
1.3 Mechanisms of antibiotic resistance Antibiotic resistance can be caused by a variety of molecular mechanisms. The resistance can be based upon antibiotic target mutation or modification, prevention of drug penetration, active efflux of antibiotics, bypass of antibiotic inhibition or enzymatic inactivation of the antibiotic substance (Blair et al., 2015).
Target mutation As many antibiotics specifically bind to their targets, a mutation of the target can lead to a decreased or prevented binding, leading to insusceptibility to the antibiotic. An example for this mechanism of resistance is the resistance to quinolones in several Gram-negative bacteria and Staphylococcus aureus. Quinolones inhibit the the bacterial enzymes DNA gyrase and topoisomerase IV that are responsible for negative supercoil introduction into the DNA (Kim & Hooper, 2014). Mutations in the gyrA and parC genes lead to changes in the active site of the enzyme, resulting in decreased inhibition by quinolones and increased resistance (Kim & Hooper, 2014).
Enzymatic target modification The resistance to an antibiotic can be based upon target modification. One example is the methylation of the ribosomal 23S subunit by the chloramphenicol-florfenicol (cfr) methytransferase. The cfr gene was first described in staphylococci, but is meanwhile found in Introduction 9 many Gram-positive and Gram-negative pathogens (Shen et al., 2013). The gene encodes for a methyltransferase which specifically methylates A2503 in the 23S rRNA, confering resistance to different classes of antibiotics that target the ribosomal 23S rRNA subunit, for example streptogamins and lincosamides (Long et al., 2006).
Enzymatic bypass The most well-known example for enzymatic bypass of antibiotic inhibition is the methicillin- resistant S. aureus -lactam antibiotics and harbours the mecA (MRSA).gene or, more This bacteriumrecently, the is mecC resistant gene. to These almost genes all β code for the alternative transpeptidase PBP2a that -lactams except ceftobiprole and ceftaroline
(Hartman & Tomasz, 1984;is Lim not & inhibited Strynadka, by 2002 β ; Bush et al., 2007; Garcia-Alvarez et al., 2011) -lactams is the inhibition of bacterial cell wall synthesis, an alternative. As thetranspeptidase mode of action can of replace β the function of the inhibited enzymes, allowing cell growth.
Reduced permeability In Gram-negative bacteria, many antibiotics have to enter the periplasm through non-specific channels, the outer membrane porins (Miller et al., 1972). By mutation or downregulation of the opr genes and by replacement of porins with more-specific channel proteins, the uptake of antibiotics into the cell can be reduced, resulting in increased resistance (Balasubramanian et al., 2011). For example, the mutation or loss of the OprD porin in Gram-negative bacteria can lead to higher resistance against the carbapenem imipenem (Sanbongi et al., 2009).
Active efflux An example for active efflux of antibiotics is the resistance to tetracyclines based on the expression of tet genes. These genes code for membrane transporters that specifically export tetracyclines and are found in both Gram-positive and Gram-negative pathogens (Kong et al., 2009). Furthermore, transporters that are able to export a wide range of antibiotics, the multidrug resistance efflux pumps, have been described. The best characterized MDR efflux pumps are the resistance nodulation division (RND) family exporters (Blair et al., 2015). RND transporters are able to confer clinically relevant levels of resistance against an extremely wide range of antibiotics (Piddock, 2006) and are found mostly in Gram-negative bacteria (Blair et al., 2015).
Enzymatic modification of antibiotics The most important mechanism of resistance in Gram-negative bacteria is the enzymatic degradation or modification of antibiotics. For example, aminoglycoside resistance is mediated Introduction 10 by production of phosphotransferases (APH), acetyltransferases (AAC) or nucleotidyltransferases (ANT) which modify the antibiotics, leading to an inactivation (Abrahams, 1941). ANTs catalyze the transfer of an AMP from an ATP molecule to a hydroxyl group in the aminoglycoside and thereby inactivate the drug. APHs transfer a phosphate residue to the aminoglycoside at different positions and are grouped into seven subgroups (Ramirez & Tolmasky, 2010). However, the most important group is the AAC group of enzymes. These enzymes catalyze the acetylation of -NH2 groups in the aminoglycoside molecule at different positions, subgrouping them into the AAC(1), AAC(3), AAC(2´) and AAC(6´) enzymes. (Ramirez & Tolmasky, 2010). However, the by far most clinically relevant example of enzymatic inactivation is the hydrolysis o -lactam antibiotics -lactamases.
f β by β 1.4 β-lactamases R -lactam antibiotics in Gram-negative bacteria can be based upon four mechanismsesistance to: i) βThe enzymatic byp -lactam-resistant alternative transpeptidase, as it the case for MRSA;ass ii)by theexpression loss of porinsof a whichβ leads to reduced outer membrane permeability; iii) the mutation of one or more PBPs and iv) the enzymatic -lactamases (Drawz & Bonomo, 2010). inactivation-lactamases by are β bacterial enzymes encoded by bla genes that can specifically bind and β -lactam antibiotics, leading to the irreversible destruction of the drug. They are the hydrolysemost common β -lactams (Livermore, 1995) and in 2015, more than 1,500 -lactamasecause ofprotein resistance sequences to β have been assigned (http://www.lahey.org/studies). uniqueThese are β distinguished by their unique 3-letter name and a number (e.g. NDM-1 for “New Delhi metallo- -lactamase 1”). The enzymes can roughly be classified by their substrate spectrum.
Narrow-β -lactamases are able to hydrolyze penicillins, while extended- - lactamasesspectrum (ESBLs) β are able to hydrolyze penicillins and cephalosporins. Carbapespectrumnemases on β the other hand are able to hydrolyze penicillins, carbapenems and mostly cephalosporins and thus can be the cause for resistance against almost all -lactam antibiotics (Cantón et al., 2012a).
However, two more detailed classification schemes β -lactamases exist. The first system is based on functional characteristics, such as preferred forsubstrate β s or inhibitor profiles. The aim of the functional classification is a correlation of enzymes to their phenotype in clinical isolates (Bush et al., 1995; Bush & Jacoby, 2010). The second scheme was developed by Ambler (1980) and is based on the amino acid sequences of the enzymes. It classifies -lactamases into molecular class A, B, C and D enzymes. This scheme is commonly used in the literatureβ and will be the one used in this study. Both systems and their characteristics are summarized in Table 1.1. The hydrolysis mechanism can be based upon two enzyme arch -lactamases of the molecular classes A, C and D possess a serine residue in their activeitectures. site that β is responsible Introduction 11
- lactamfor an nucleophilicring (Figure attack1.4). This of theresults hydroxyl in the group formation of the of serinea covalent on the acyl carbonyl ester. Hydrolysis group of theof the β ester utilizing a catalytic water molecule finally leads to the separation of the complex, leaving the -lactam (Livermore, 1995; Drawz & Bonomo,
2010intact). With and the active formation enzyme of and a covalenty the inactivated bound βacyl enzyme, the mechanism is similar to the -lactams (Ghuysen, 1991). In contrast to PBPs, where the hydrolysis is of inhibition of PBPs by β -lactamases is suchvery effa lowicient rate and that the it complexeffectively dissociates leads to an quickly inhibition, after thesucessful hydrolysis hydrolysis by serine (Livermore, β 1995).
Table 1.1 Classification schemes for β-lactamases according to Bush & Jacoby (2010) and Ambler (1980). Table obtained and modified from Bush & Jacoby (2010). The table is sorted according to the Bush/Jaboby scheme, although this scheme will not be used in this study.
Group Molecular Inhibited by Distinctive Represantative (Bush & class CA or Defining characteristics substrate(s) EDTA enzyme(s) Jacoby) (Ambler) TZBa Greater hydrolysis of E. coli AmpC, ACT-1, cephalosporins than 1 C Cephalosporins No No CMY-2, FOX-1, MIR- benzylpenicillins, hydrolyzes 1 cephamycins Increased hydrolysis of 1e C Cephalosporins No No ceftazidime and often other GC1, CMY-37 oxyimino- -lactams Greater hydrolysis of 2a A Penicillins Yes No benzylpenicillinβ than PC1 cephalosporins Similar hydrolysis of Penicillins, early TEM-1, TEM-2, SHV- 2b A Yes No benzylpenicillin and cephalosporins 1 cephalosporins Increased hydrolysis of oxyimino- Extended-spectrum -lactams (cefotaxime, TEM-3, SHV-2, CTX- 2be A Yes No cephalosporins ceftazidime, ceftriaxone, cefepime, M-15, PER-1, VEB-1 aztreonam)β Resistance to clavulanic acid, 2br A Penicillins No No TEM-30, SHV-10 sulbactam and tazobactam Increased hyrolysis of oxyimino- - Extended-spectrum lactams combined with resistance 2ber A cephalosporins, No No TEM-50 to clavulanic acid, sulbactam andβ monobactams tazobactam Increased hydrolysis of 2c A Carbenicillin Yes No PSE-1, CARB-3 carbenicillin Increased hydrolysis of Carbenicillin, 2ce A Yes No carbenicillin, cefepime and RTG-4 cefepime cefpirome Increased hydrolysis of cloxacillin 2d D Cloxacillin Variable No OXA-1, OXA-10 or oxacillin Extended-spectrum Hydrolyzes cloxacillin or oxacillin 2de D Variable No OXA-11, OXA-15 cephalosporins and oxyimino- -lactams Hydrolyzes cloxacillin or oxacillin 2df D Carbapenems Variable No OXA-23, OXA-48 and carbapenemsβ Hydrolyzes cephalosporins. Extended-spectrum 2e A Yes No Inhibited by clavulanic acid but CepA cephalosporins not aztreonam Increased hydrolysis of 2f A Carbapenems Variable No carbapenems, oxyimino- - KPC-2, IMI-1, SME-1 lactams, cephamycins β IMP-1, VIM-1, CcrA, B (B1) Broad-spectrum hydrolysis IND-1, NDM-1 3a Carbapenems No Yes including carbapenems, but not L1, CAU-1, GOB-1, B (B2) monobactams FEZ-1 Preferential hydrolysis of 3b B (B2) Carbapenems No Yes CphA, Sfh-1 carbapenems a CA, clavulanic acid; TZB, tazobactam Introduction 12
Figure 1.4 Action of a serine β-lactamase against carbapenems. -lactam ring is attacked by the free hydroxyl of the enzymes active site serine residue, yielding a covalent azyl ester. Hydrolysis of the ester with the help of a catalytic water molecule finally leads to the dissociationAfter of thebinding, comple the β -lactam antibiotic is irreversibly inactivated. Figure obtained and modified from Wilson et al. (2010). x and the β In contrast to serine- -lactamases, metallo- -lactamases (MBL) utilize one or two zinc ions that coordinate a water moleculeβ which β -lactams’ amide bond. In is used -forlactam the (attackDrawz on& Bonomo, the β 2010). addition, MBLs do not covalently bind to the β 1.4.1 Class A β-lactamases TEM- -lactamase, was identified in 1965. It was the first plasmid-
1, the -lactamase first class described A serine βand nowadays, TEM-type enzymes, together with SHV- - lactamasesmediated β are frequently found in Gram-negative clinical isolates (Drawz & Bonomo, 2010type). βIn the early 1980s, shortly after the introduction of extended-spectrum cephalosporins cefotaxime and ceftazidime, the first class A ESBLs were identified that conferred resistance against these antibiotics (Drawz & Bonomo, 2010). Today, enzymes of the CTX-M type are the most important class A ESBLs, as the encoding genes are often located on highly transmissible plasmids that spread into a wide range of Gram-negative pathogens (Bonnet, 2004; Drawz & Bonomo, 2010). Although these enzymes are able to hydrolyze penicillins, narrow- and extended-spectrum -lactamase cephalosporinsinhibitors sulbactam, and aztreonam, tazobactam they and areclavulanic inhibited acid by (Drawz the commercially & Bonomo, 2010 available). In contrast, β the -lactams, including carbapenems and classmonobactams, A carbapenemases but are still are inhibited able to by hydrolyze the mentioned all β substances (Bonnet, 2004). The most important class A carbapenemases are NMC/IMI, SME and KPC-type enzymes and certain GES variants (Diene & Rolain, 2014). All enzymes of this class share a highly conserved STKF motif at -lactamase standard numbering thescheme amino with acid the positions Ser70 residue 70 to 73 according to the class A β -lactam ring (Ambler et al., 1991). Al beeing the active site- lactamaseserine that genes covalently are fo bindsund onthe plasmids,β several chromosomally locatedthough or integron most class-bourne A β class A genes (e.g. GES-1) have been described (Drawz & Bonomo, 2010).
Introduction 13
1.4.2 Class B β-lactamases -lactamases, or metallo- -lactamases (MBLs), differ substantially from the other classes.Class B Insteadβ of an active site serineβ the hydrolysis mechanism uses one or two zinc ions that are coordinated in the active site of the enzyme (Gupta, 2008a). By coordination of a water molecule by the zinc ions and the use of the -OH group of the water the enzyme performs the -lactam substrate, resulting in an opening of the ring
(hydrolyticDrawz & attackBonomo, on the2010 amide). Because bond of ofthe theirβ unique hydrolysis mechanism, MBLs are not inhibited by clinically available inhibitors like sulbactam, clavulanic acid or tazobactam. In vitro, MBLs can be inhibited by EDTA, which chelates the zinc ions that are necessary for hydrolysis, -lactamase (Drawz & Bonomo, 2010). In contrast to the class A,
Cmaking and D them enzymes unavailable that belong to the to β the acyltransferases of the SxxK superfamily, MBLs belong to their own superfamily, also including enzymes with non- -lactamase functions (Cornaglia et al.,
2011). β The substrate spectrum of MBLs differs between the numerous enzyme variants. For example, the CphA metallo- -lactamase of Aeromonas hydrophila has a rather narrow substrate spectrum while extended rangeβ enzymes like the VIM- or IMP- - lactams, including carbapenems, but sparing monobactamstype MBLs(Cornaglia are able et al. to, 2011 hydrolyze). MBLs all are β subcategorized into three subclasses. The B1 subclass enzymes require at least one zinc ion in their active site to be fully active. The most clinically relevant members of this subclass are the VIM, IMP and NDM enzymes (Nordmann & Poirel, 2014). The B2 enzymes, for example CphA, require only a single zinc ion and are even inhibidted by a second one, while the B3 MBLs essentially require two zinc ions, for example the L1 MBL from Stenotrophomonas maltophilia (Cornaglia et al., 2011). -lactam by the carboxylate and carbonyl groups.L1 and After other binding, dicationic the carbonyl enzymes is coordinate polarized by the one β of the zinc ions and attacked by the -OH group of a water molecule. This leads to an anionic state of the nitrogen -lactam, which is than protonated, leaving the o -lactam ring. The source of this inproton the β is still unknown. For B2 enzymes it is proposedpened thatβ the water molecule is not coordinated by the single zinc ion, but by the enzyme residues His118 or Asp120 and that the zinc ion is responsible for coordinat -lactam nitrogen (Drawz & Bonomo, 2010). The zinc binding ligands are highly consionerved of thebetween β the members of each subclass. Among the most clinical relevant subclass B1 enzymes, the first zinc ion is bound by the amino acid residues His116, His118 and His196, while the second one binds to the residues Asp120, Cys221 and His263, follow -lactamases standard numbering scheme (Garau et al., 2004).
MBL encoding inggenes the can Class be Bchromosomally β located (e.g. L1 from S. maltophilia) or plasmid- bourne like blaVIM or blaNDM and are often found within integron structures (Cornaglia et al., 2011).
Introduction 14
1.4.3 Class C β-lactamases -lactamases, or AmpC enzymes, are serine- -lactamases. In 1940, the E. coli AmpC wasThe classthe first C β enzyme reported to inactivate penicillin (Abrahamβ & Chain, 1940). The most AmpC enconding genes are located on the bacterial chromosome, but plasmid-bourne AmpC enzymes are becoming more prevalent (Drawz & Bonomo, 2010). AmpC genes can be found in many Enterobacteriaceae like Enterobacter spp., Citrobacter freundii or E. coli and in P. aeruginosa or A. baumannii, while Klebsiella spp., Salmonella spp. and Proteus spp. normally do not harbour chromosomal AmpC encoding genes (Jacoby, 2009). In most cases, the expression level of blaAmpC genes is rather low, but in some species can be induced by exposure to -lactams, especially cefoxitin and imipenem (Bennett & Chopra, 1993; Babic et al., 2006certain). The βinduction mechanism is based on the conformational change of the transcriptional regulator AmpR that is induced by binding of cell wall fragments that are formed -lactam treatment. This has an
-lactamsunder β can become resistant during importanttherapy ( Jacoby, clinical 2009 impact,; Drawz as strains & Bonomo, susceptible 2010 to ). β In addition, AmpCs are sometimes overexpressed in clinical isolates, resulting from mutations in the ampD or ampC genes that lead to hyperinducibility or to constitutive expression (Jacoby, 2009). Although carbapenems are hydrolyzed with only weak activity, an AmpC overexpression combined with a porin loss and efflux systems can lead to increased carbapenem resistance in clinical P. aeruginosa isolates (Jacoby, 2009). Examples for AmpC enzymes are CMY-2, ACT-1, DHA-1 and the E. coli AmpC (Bush & Jacoby, 2010).
1.4.4 Class D β-lactamases With currently over 450 variants assigned, class D serine -lactamases are one of the largest group -lactam hydrolyzing enzymes. They are also knownβ as OXA-type enzymes, named after their initialof β characteristic: the ability to hydrolyze oxacillin with higher efficiencies than class A -lactamases (Drawz & Bonomo, 2010). They display very low levels of homology to Class A and
β -lactamases (Massova & Mobashery, 1998) and are a very heterogenous group of enzymes Cthat β is found in a wide variety of Gram-negative bacteria with clinical importance. They were mostly identified in P. aeruginosa, E. coli, P. mirabilis and A. baumannii isolates (Leonard et al., 2013). OXA- -lactamase genes are characterized as highly mobile, as most of them have been found ontype plasmids, β in transposons or within mobile integrons (Poirel et al., 2010). Contrary to mobile blaOXA genes, it was found that every A. baumannii strain intrinsically harbours the chromosomally encoded OXA-51 -lactamase (Evans & Amyes, 2014).
While many OXA-type enzymes areβ described as narrow- -lactamases or ESBLs (e.g. OXA-2, OXA-10 and OXA-20), the class also harbours spectrumcarbapenemases β that are known as carbapenem- -lactamases (CHDLs) with OXA-48 beeing the most prominent and clinicallyhydrolyzing relevant one class (Poirel D β et al., 2010; Leonard et al., 2013). -lactamases can OXA β Introduction 15 significantly differ from each other with homologies of only 30 % and the enzymes are subgrouped, for example into the OXA-2, OXA-10 and OXA-23-like enzymes (Evans & Amyes, 2014). Despite their great difference, OXA enzymes share several highly conserved regions, with one of them beeing the region around the serine amino acid residue at position 70, relative to -lactamase numbering system (De Luca et al., 2011). This residue is part of the theSTF class D β -lactam substrate.K motif The (positions two other 70 hig to hly73) conserved and is the regions active siteare theserine YG Nthat motif covalently at the positions binds the 144 β to 146 and the KTG motif at the positions 216 to 218. These motifs are found in almost all OXA enzymes (Poirel et al., 2010).
1.5 Carbapenemases and their distribution As previously described, carbapenemases are found in the molecular classes A, B and D. Although these enzymes differ in their hydrolytic efficiency against v -lactam substrates, they are often conferring high level resistance to carbapenems in clinicalarious Gram β -negative isolates (Queenan & Bush, 2007). The carbapenemases of the Ambler class A are the IMI/NMC, SME, KPC and GES-type enzymes (Diene & Rolain, 2014). GES-1 has been described as an ESBL, but novel variants of this enzyme like GES-2 or GES-5 have been found that exhibited significant carbapenem hydrolysis (Nordmann et al., 2012). SME, IMI and NMC enzymes are usually chromosomally encoded, whereas GES and KPC enzymes are plasmid-encoded (Diene & Rolain, 2014). The currently clinically most relevant class A carbapenemase is KPC-2, which was originally identified in a K. pneumoniae isolate in the U.S. in 1996 but is nowadays found in many Gram-negative species and has spread globally within a few years (Nordmann & Poirel, 2014). All class B metallo- -lactamases are classified as carbapenemases. While MBLs are intrinsic for many environmentalβ and opportunistic bacterial species, several acquired mobile MBLs have been identified since the early 1990s (Walsh et al., 2005). They were mostly found in clinical P. aeruginosa strains or in Enterobacteriaceae (Nordmann et al., 2012). The most common MBLs belong to the IMP, VIM and NDM type, but also other types have been described that are found less frequent, for example GIM, KHM, FIM and SIM (Queenan & Bush, 2007; Sekiguchi et al., 2008; Pollini et al., 2013; Diene & Rolain, 2014; Nordmann & Poirel, 2014). MBL genes can be located on conjugable plasmids or mobile transposons and are distributed worldwide with several regional accumulations (Diene & Rolain, 2014). In many cases, MBL genes are found within integron structures or as part of larger transposons (Walsh et al., 2005; Cornaglia et al., 2011). Currently, VIM-2 is the most reported MBL wordwide and is mostly found in southern Europe (Greece, Spain and Italy) and in South Korea and Taiwan (Nordmann & Poirel, 2014). NDM enzymes on the other hand are mostly found on the Indian subcontinent (India, Pakistan and Sri Lanka) but have also rapidly spread worldwide since their first description in 2009 and NDM-1 is currently one of the most clinically relevant carbapenemases (Yong et al., 2009; Introduction 16
Nordmann & Poirel, 2014). The third important group of MBLs are the IMP-type enzymes. IMP-type MBLs were the first acquired MBLs to be identified in 1991 and have spread into many Gram-negative species with clinical importance since then (Cornaglia et al., 2011). So far, 50 IMP variants have been assigned (http://www.lahey.org/studies) and these enzymes have spread worldwide, mostly in P. aeruginosa and A. baumannii strains (Nordmann & Poirel, 2014). Class D carbapenemases, or CHDLs, can be plasmid- or chromosomally encoded (Diene & Rolain, 2014). The clinically most relevant OXA carbapenemase is the plasmid-encoded OXA-48, which has been primarily found in Enterobacteriaceae. It was first described in K. pneumoniae in 2003 and has spread widely since. OXA-48 is mainly found in Turkey and most other countries of the Mediterranean area, but is also frequently found in nearly all European countries and Northern Africa (Diene & Rolain, 2014; Nordmann & Poirel, 2014). Other important OXA-type carbapenemases are the OXA-23-, OXA-24- and OXA-58-like enzymes which are found worldwide and mainly in A. baumannii isolates (Walsh, 2010). They can be chromosome- or plasmid-encoded (Evans & Amyes, 2014).
1.6 Mobility of β-lactamase genes -lactamase genes or resistance genes in general can be transferred between bacteria with variousβ mechanisms. The two clinically most important mechanisms that mediate this horizontal gene transfer are conjugative transposable elements and conjugable plasmids (Diene & Rolain, 2014). Conjugable transposable elements are genetic structures that encode all functions necessary for their own intercellular transfer and are subgrouped into conjugable transposons (Tn) and insertion sequences (IS) (Siguier et al., 2014).
Insertion sequences Insertion sequences (IS) are relatively small DNA structures (0.7 to 2.5 kb) that carry one or two open reading frames (ORFs) that encode for transposases. Transposases are multifunctional enzymes that catalyze the excision and the transfer of DNA sequences (Siguier et al., 2014). IS are bordered by short terminal inverted repeat sequences that function as recognition sites for the transposase (Darmon & Leach, 2014). ISs can jump into the chromsome as well as into plasmids (Siguier et al., 2014). Although a classical IS does not harbour additional genes, many IS families are more complex and can carry passenger genes that encode for regulatory proteins, methyltransferases or antibiotic resistance (Figure 1.5). They are known as transporter ISs (Siguier et al., 2014) -lactamase genes. For example, .the IS elementsblaOXA-48 gene have is frequently almost always been flanked reported by as one carriers or two for copies β of the insertion sequence IS1999 (Evans & Amyes, 2014).
Introduction 17
Figure 1.5 Schematic organization of transporter insertion sequences and transposons. (A) Organization of a typical transporter IS. The IS is flanked by two short inverted repeat regions (IRL and IRR) that encompass one or two transposase encoding genes and one or more passenger genes. When the IS is inserted, a short sequence of the target DNA is often duplicated, resulting in direct repeats (DR) that encompass the IS. (B) Organization of a typical transposon. The transposon is flanked by larger inverted repeat regions and carries multiple genes that are responsible for transposition. It can also carry additional accessory genes that can be resistance genes or other genes conferring a phenotypical advantage to the host cell. Figure obtained and modified from Darmon & Leach (2014).
Transposons Transposons are large DNA structures with sizes ranging from 2.5 to 60 kb (Darmon & Leach, 2014) and encode for site-specific DNA recombinases that function as integrases, resolvases and invertases (Burrus et al., 2002). These enzymes catalyze the integration and excision of DNA, the resolution of co-integrates and the inversion of DNA fragments (Darmon & Leach, 2014). Like ISs, transposons can be integrated into the chromosome or into plasmids (Darmon & Leach, 2014). Transposons usually possess long terminal inverted repeats and often harbour accessory genes that confer an phenotypic advantage to their host, for example antibiotic or heavy metal resistance genes (Darmon & Leach, 2014). The structure of a typical transposon is illustrated in Figure 1.5. Complex conjugative transposons are called composite transposons, that possess ISs at both ends and can excise themselves for conjugation to another cell (Darmon & Leach, 2014). -lactamase gene carrying transposons have been described, for example
TnA large4401 that number carries of βthe blaKPC-2 gene (Cuzon et al., 2010). Tn2006 in A. baumannii is carrying the blaOXA-23 gene and is almost allways a composite transposon that is bracketed on both sides by the insertion sequence ISAbaI (Diene & Rolain, 2014). ISAba1 has also been reported as a carrier for blaOXA-51-like, blaOXA-58-like and blaOXA-235-like genes (Evans & Amyes, 2014).
Plasmids Self-transmissible conjugative plasmids are large DNA molecules that encode the proteins involved in their own transfer from a donor cell to a recipient cell via conjugation. They exist separately from the bacterial chromsome and are replicated independently from it, although the replication infrastructure is mainly provided by the host cell (Bennett, 2008). The size of plasmids ranges between a few thousand to hundreds of thousands of base pairs and in most cases, they are circular molecules, although linear plasmids exist, for example in Introduction 18
Streptomyces spp. or Borellia burgdorferi (Snyder & Champness, 2007). All conjugable plasmids exhibit two important regions, the oriV and oriT regions. The oriV (V for vegetative) region is the origin of replication and is the main determinant for the plasmid host range and the copy number regulation, although conjugative plasmids are mostly single copy molecules (Snyder & Champness, 2007). Another important function of the oriV is determination of the incompatibility type, which is a regulative mechanism that determines the stable coexistance of two or more plasmids in one cell. If two plasmids cannot coexist stably in the cell, they share the same incompatibility (Inc) type (Snyder & Champness, 2007). The oriT (T for transfer) is the origin of the rolling-circle replication during conjugation (Snyder & Champness, 2007). The genes necessary for transfer are the tra genes, which occur in various combinations and are correlated to the plasmids Inc-type (Snyder & Champness, 2007). Usually, plasmids carry genes that confer a growth advantage for the host cell. These can be resistance determinants and since the first detection of antibiotic resistance, plasmids have been the major distributives of antimicrobial resistance genes (Bennett, 2008). Many important carbapenemase genes are plasmid-mediated, for example OXA-48, NDM-1, KPC-2, and VIM-1 and in many cases, the genes are part of integrons (Smith Moland et al., 2003; Poirel et al., 2004b; Loli et al., 2006; Johnson & Woodford, 2013).
Integrons Integrons are genetic structures that efficiently capture and express genes. They are often part of larger insertion sequences or transposons and thus can be mobilized (Mazel, 2006). The structure of integrons is characterized by several core features. The first feature is the intI gene encoding for an integrase, which catalyzes the recombination between incoming gene cassettes and the second core feature, the attI site. This site is an integron-associated recombination site. The third core feature is the expression of captured genes by one or two integron-associated promoters (Gillings, 2014). Novel genes are acquired by insertion of circular gene cassettes, which usually consist of a single ORF and the attC element (Hall et al., 1991). The gene is inserted by site-specific recombination between the attI and attC sites and this process is catalyzed by the integrase (Gillings, 2014). While integrons were classified into five groups at first, it is nowadays known that hundreds of different integron classes exist, based on their respective intI sequences (Boucher et al., 2007). The most clinically relevant classes of integrons are the classes 1, 2 and 3, which are all linked to insertion sequences and transposons, conferring a mobility of the integrated gene cassettes. The most frequently found integrons that are associated with antibiotic resistance genes are the class 1 integrons (Mazel, 2006). The structure of a typical class 1 integron is shown in Figure 1.6. Class 1 integrons consist of two highly conserved regions, the 5´CS region, which includes the intI1 gene and the attI site and the
Introduction 19
Figure 1.6 Schematic structure of a class 1 integron. The conserved integron regions consist of the integrase- encoding intI1 gene and the qacEΔ1/sul1 open reading frame. Resistance gene cassettes (gene + attC site) can be acquired and inserted at the attI site and are expressed under the control of the promoters Pc and P2.
3´CS region, including the partially deleted gene qacEΔ1 and the sul1 gene that confer resistance against quarternary ammonium compounds and sulfonamides (Mazel, 2006). They possess three promoter structures that are Pint, Pc and P2 (Collis & Hall, 1995). Pint is the promoter of the intI1 gene, while Pc and P2 control the expression of the integrated genes cassettes and can occur in several variations, resulting in different expression levels (Papagiannitsis et al., 2009). Class 1 integrons have been described as carriers of blaIMP, blaVIM, blaOXA and aac-type genes and play an important role for the dissemination of antibiotic resistance (Walsh et al., 2005; Voulgari et al., 2013).
1.7 Pseudomonas aeruginosa P. aeruginosa is a Gram-negative opportunistic pathogen that can cause a wide range of severe nosocomial infections. It normally inhabits the soil and surface in aqueous environments and exhibits several intrinsic antibiotic resistance determinants, such as low permeability, -lactamase (Gellatly & Hancock, 2013).
P.expression aeruginosa of effluxis one systems of the andmost an induciblecommon AmpCpathogens β causing respiratory infections in hospitalized patients. In almost all cases infections occur only in patients with poor health status and as most clinical P. aeruginosa strains carry multiple resistance genes in addition to their intrinsic resistance mechanisms isolates are often multidrug resistant. Consequently, morbidity and mortality based on P. aeruginosa infections are rather high (Gellatly & Hancock, 2013). Apart from pneumonia, the bacterium is also capable of infecting the urinary tract and soft tissue (e.g. after burns) and can cause bacteremia, keratitis and other infections (Gellatly & Hancock, 2013). P. aeruginosa strains have frequently been described as carriers of carbapenemases, with the majority beeing MBLs of the IMP, VIM, NDM and GIM families (Diene & Rolain, 2014).
1.8 Citrobacter freundii Like P. aeruginosa, C. freundii is a Gram-negative opportunistic pathogen and can cause severe nosocomial infections in neonates or immunocompromised adults or older children (Doran, 1999). As a member of the Enterobacteriaceae, C. freundii belongs to the resident commensal flora of the human gastrointestinal tract, although it is assumed that Citrobacter species have a wide environmental distribution (Janda & Abbot, 2006). It can cause infections of the central Introduction 20 nervous system, bacteremia and urinary tract infections. Rarely, C. freundii strains are described in the context of wound infections, respiratory tract infections and gastroenteritis cases (Janda & Abbot, 2006). -lactamases is common in Citrobacter species and C. freundii always harboursExpression a chromosomally of β -encoded ampC gene. It has also been described as a carrier for several plasmid-encoded ESBLs like CTX-M-3 or carbapenemases of the KPC, OXA-48 and IMP type (Janda & Abbot, 2006; Diene & Rolain, 2014).
1.9 Objectives of this work At the German National Reference Laboratory for multidrug-resistant Gram-negative bacteria at the Ruhr-University Bochum, Gram-negative clinical isolates with increased carbapenem resistance are analyzed for the molecular basis of resistance. The resistance is analyzed with phenotypic and genetic methods, including several antibiotic disk-based tests and a PCR screening on the most common carbapenemase genes, but also on genes that are less frequent. In some clinical isolates, the cause of resistance can only be identified phenotypically and there is a chance that these isolates harbour novel resistance genes or variants of existing ones. As it has been shown that already one single amino acid substitution can significantly change the -lactamase, the identification and characterization of these enzymeshydrolysis is important characteristics for bo ofth aclinical β diagnostics and antimicrobial therapy.
In this study, three clinical isolates were analyzed on the molecular basis of carbapenem resistance: P. aeruginosa NRZ-00156, C. freundii NRZ-02127 and P. aeruginosa NRZ-03096. P. aeruginosa NRZ-00156 was isolated in 2008 from an ingunial swab from a patient hospitalized in Western Germany and showed high carbapenem resistance and a clear carbapenemase phenotype. This phenotype was inhibited by EDTA, indicating the potential production of a metallo- -lactamase. However, all diagnostic PCRs for MBL genes were negative and it was suspected thatβ this isolate harboured a novel MBL gene. C. freundii NRZ-02127 was isolated in 2011 from tracheal aspirate from a patient hospitalized in Southern Germany and showed elevated carbapenem resistance but was susceptible to oxyimino-cephalosporins. It also showed a carbapenemase phenotype that was inhibited by clavulanic acid. It was suspected that this isolate har -lactamase, although the resistance to carbapenems and the inhibition by clavulanicboured acid a class did Dnot β match to any described OXA-type enzyme and diagnostic PCRs for blaOXA-48-like genes were negative. P. aeruginosa NRZ-03096 was isolated in 2012 from an anal swab from a patient hospitalized in Northern Germany and also showed high carbapenem resistance and an MBL phenotype, as the resistance was inhibited by EDTA. Like in P. aeruginosa NRZ-00156, diagnostic PCRs covering all common MBL genes were negative and it was suspected that this isolate produced a novel carbapenemase. Introduction 21
The main objectives that were adressed in this work are: i) the search for novel carbapenemases; ii) the characterization of the genetic environment of the carbapenemase genes and their localization; iii) the phenotypic characterization of the enzymes and their impact on resistance in vivo and iv) the biochemical characterization of the novel enzymes. The identification of novel carbapenemases was adressed by PCR and shotgun cloning approaches and phenotypic characterization by resistance analyses in isogenic E. coli strains. The characterization of the genetic environment was adressed by sequencing and in silico DNA sequence analysis techniques, while the localization of the genes was analyzed by Southern blotting experiments and 454-sequencing of plasmids. The biochemical characterization was adressed by overexpression and purification of the native unmodified novel enzymes and by obtaining the kinetic parameters Km and kcat for the most important -lactam substrates with in vitro hydrolysis assays.
β These experiments were performed to make a statement on the capabilities of novel carbapenemases, their ongoing diversification, their ability to spread and their potential significance for future carbapenem resistance developments in Gram-negative bacteria of clinical importance. Material and Methods 22
2 Material and Methods
All instruments or materials that were used in this study but not listed here were standard lab equipment. All solutions and lab materials for bacterial cell cultures and molecular techniques were autoclaved or filtered sterile before use. All buffers and media were prepared in A. dest except noted otherwise.
2.1 Material 2.1.1 Instruments Cell disruptors: PowerLyzer 24 MO BIO Sonifier W-250 D Branson Chromatography instruments: ÄKTA Pure 25 L FPLC system GE Healthcare HiPrep 16/60 S-200 HR GF column GE Healthcare HiPrep 26/10 desalting column GE Healthcare HiTrap SP HP 5 ml IEX column GE Healthcare Centrifuges & rotors: Centrifuge 3K12, rotor #11223 Sigma-Aldrich Biofuge Pico, rotor #3325 Thermo Scientific-Heraeus Megafuge 1.0R, rotor #3360 Heraeus Fresco 21, rotor #75003424 Thermo Scientific-Heraeus J2-HS, rotors JA-14 & JA-20 Beckmann Electrophoresis instruments: CHEF DR III PFGE chamber Bio-Rad DNA gel electrophoresis chamber Renner Multiphor II IEF chamber GE Healthcare SDS -PAGE chamber P8DS Thermo Scientific / Owl Scientific GelDoc XR+ gel documentation system Bio-Rad HyperCassette exposure cassette Amersham Incubators: Incubator BB 16 Heraeus Instruments Incubator shaker 3033 GFL innova 4330 New Brunswick Scientific PureLab Classic water destiller Elga Material and Methods 23
Next-generation sequencing instruments: DNA nebulizer Roche DynaMag magnetic particle concentrator Invitrogen GS Junior 454 sequencing system Roche GS Junior Bead Counter Roche GS Junior Titanium PicoTiterPlater Roche PCR cyclers: Labcycler basic Sensoquest Labcycler gradient Sensoquest Mastercycler personal Eppendorf Mastercycler epgradient Eppendorf pH-Meter 761 Calimatic Knick Photometers and accessories: BioPhotometer Eppendorf BioSpectrometer kinetic Eppendorf Eppendorf µCuvette G1.0 Eppendorf Power supply: Gene Power Supply 200/400 Pharmacia Power Pac 300 Power Supply BioRad Qubit 2.0 fluorometer Life Technologies Thermomixers: ThermoMixer C Eppendorf TS1 Thermoshaker Biometra Vacu-Blot Southern blotting chamber Biometra VITEK Densichek densitometer bioMérieux
2.1.2 Disposable material CleanGel IEF GE Healthcare Sterile cotton tips Heinz Herenz Hamburg CryoPure cryotubes Sarstedt Disposable PFGE Plug Mold Bio-Rad Filtropur S 0.2 sterile filter Sarstedt Fitropur S 0.45 sterile filter Sarstedt Gel blot paper Schleicher & Schüll Glass beads 0.1 mM Scientific Industries HyperFilm MP autoradiography film GE Healthcare MH2 Mueller-Hinton agar plates bioMérieux Material and Methods 24
Nylon membrane, positively charged Roche UVette cuvettes Eppendorf UV-transparent cuvettes Sarstedt
2.1.3 Chemicals 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) AppliChem -Mercaptoethanol AppliChem
βAcetic acid p.a. VWR Acrylamide (30%) Rotiphorese Gel 30 Roth AEBSF-hydrochloride AppliChem Ammonium persulfate (APS) Roth Bovine serum albumine (BSA) Fraktion V AppliChem Brilliant Blue R-250 Roth Bromophenole blue Merck Calcium chloride J.T. Baker Clavulanic acid SmithKline-Beecham Chloro-5-substituted adamantyl-1,2- dioxetane phosphate (CSPD) Roche Defibrinated sheep blood Thermo Scientific Dimethyl sulfoxide (DMSO) Biomol DNA-Polymerasepuffer GE Healthcare Deoxyribose nucleoside triphosphates (dNTPs) Applied Biosystems, Thermo Scientific Dipotassium phosphate Merck Disodium phosphate J.T. Baker Ethanol p.a. Sigma-Aldrich Ethylenediaminetetraacetic acid (EDTA) Merck Ethidiumbromide AppliChem Formamide J.T. Baker Glycerol J.T. Baker Hydrogen chloride 36-38% J.T. Baker InCert agarose Lonza Isopropyl alcohol J.T. Baker Magnesium chloride J.T. Baker Magnesium sulfate J.T. Baker Maleic acid Merck Material and Methods 25
Manganese(II) chloride Merck Methanol p.a. J.T. Baker Monopotassium phosphate Riedel-de Haën Monosodium phosphate J.T. Baker Nitrocefin Calbiochem Petroleum Roth Polyoxyethylene (20) cetyl ether (Brij 58) Sigma-Aldrich Roti-Nanoquant Roth Rubidium chloride AppliChem Seakem Gold Agarose Lonza Sodium dodecyl sulfate AppliChem, Sigma Sodium hydroxide J.T. Baker Sodium chloride J.T. Baker Sodium citrate Merck Sodium lauroyl sarcosinate Sigma-Aldrich StarPure agarose StarLab Sucrose AppliChem Tetramethylethylenediamine (TEMED) Merck Tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) AppliChem Tris(hydroxymethyl)aminomethane (Trizma base) Sigma-Aldrich Tween20 AppliChem Zinc sulfate AppliChem
2.1.4 Antibiotics Penicillin G Molekula Ampicillin Molekula, AppliChem Oxacillin Molekula Piperacillin Molekula Cefotaxime Molekula, Aventis Ceftazidime Molekula Cefoxitin Molekula, Infektiopharm Imipenem Molekula Ertapenem Molekula, MSD Meropenem Molekula, Hexal Aztreonam Molekula Kanamycin AppliChem Chloramphenicol AppliChem Material and Methods 26
Sodium azide Riedel-de Haën Rifampicin AppliChem Streptomycin AppliChem
2.1.5 Wafers containing antibiotics Susceptibility disks Thermo Scientific Oxoid
Ampicillin 10 µg, Ampicillin/Sulbactam 10/10 µg, Piperacillin 30 µg, Piperacillin/Tazobactam 36 µg, Amoxicillin 10 µg, Amoxicillin/Clavulanic acid 30 µg, Cefotaxim 5 µg, Cefoxitin 30 µg, Imipenem 10 µg, Meropenem 10 µg, Doripenem 10 µg, Ertapenem 10 µg
2.1.6 Antibiotic gradient test strips Etest bioMérieux
Ampicillin, Ampicillin/Sulbactam, Piperacillin, Piperacillin/Tazobactam, Amoxicillin, Amoxicillin/Clavulanic acid, Temocillin, Cephalothine, Cefuroxime, Cefoxitin, Cefotaxime, Ceftriaxon, Cefepime, Ceftazidime, Imipenem, Meropenem, Doripenem, Ertapenem, Aztreonam, Gentamicin, Tobramicin, Amikacin, Doxycyclin, Tetracyclin, Minocyclin, Tigecyclin, Ciprofloxacin, Levofloxacin, Colistin, Nitrofurantoin, Chloramphenicol, Fosfomycin, Trimethoprim/Sulfamethoxazole, MBL Etest Imipenem, MBL Etest Meropenem
2.1.7 Kits und standards Kits: Expand Long Range PCR System Roche FastStart High Fidelity PCR System Roche GS Junior Titanium emPCR Kit (Lib-L) Roche GS Junior Maintenance Wash Kit Roche GS Junior Titanium PicoTiterPlate Kit Roche GS Junior Titanium Sequencing Kit Roche GS Rapid Library Kit Roche Nucleospin Tissue Kit Macherey-Nagel Nucleospin Plasmid Kit Macherey-Nagel Nucleospin Gel and PCR cleanup Kit Macherey-Nagel NucleoBond PC 100 Kit Macherey-Nagel PCR DIG Probe Synthesis Kit Roche Qubit dsDNA HS Assay Kit Life Technologies Qubit Protein Assay Kit Life Technologies Material and Methods 27
Universal GenomeWalker 2.0 Kit Clontech Standards: CHEF DNA Size Marker #170-3605 Bio-Rad CHEF DNA Size Marker #170-3667 Bio-Rad GeneRuler DNA Ladder Mix Thermo Scientific Lambda-Ladder PFG marker New England Biolabs Low range PFG marker New England Biolabs PageRuler Plus Prestained Thermo Scientific
2.1.8 Enzymes FastDigest DNA restriction endonucleases Thermo Scientific I-Ceu I restriction endonuclease New England Biolabs Lysozyme AppliChem Nuclease S1 restriction endonuclease Thermo Scientific Pfu proof-reading DNA Polymerase Thermo Scientific Proteinase K Boehringer Mannheim Pwo SuperYield DNA Polymerase Roche RNase A AppliChem T4 DNA Ligase Thermo Scientific, Roche Taq DNA Polymerase Roche, Peqlab
All enzyme reactions were performed within the appropriate buffer supplied by the manufacturer.
2.1.9 Antibodies Anti-Digoxigenin-AP, Fab fragments Roche
Material and Methods 28
2.2 Microbial strains, plasmids and oligonuclotides
2.2.1 Microbial strains All microbial strains used in this study are listed in Table 2.1.
Table 2.1. Microbial strains used in this study.
Strain Relevant characteristics Reference / Source E. coli TOP10 Cloning host, lacI- Invitrogen
E. coli J53 NaN3R Clowes & Rowley (1954) E. coli C600 + RifR RifR RUB Control strain for various resistance E. coli ATCC 25922 tests LCG Standards P. aeruginosa NRZ-00156 Clinical isolate RUB C. freundii NRZ-02127 Clinical isolate RUB P. aeruginosa NRZ-03096 Clinical isolate RUB
2.2.2 Plasmids All plasmids used or constructed in this study are listed in Table 2.2.
Table 2.2. Plasmids used in this study.
Plasmid Relevant characteristics Reference / Source pBK-CMV Cloning & expression vector, KmR Invitrogen pBK-CMV derivative carrying a pMB3002 gDNA fragment from C. freundii Meining (2012) NRZ-02127 pBK-CMV derivative carrying the pMB3006 Meining (2012) blaOXA-233 gene pBK-CMV derivative carrying the pMB3007 This study blaIMP-31 gene pBK-CMV derivative carrying the pMB3010 This study blaIMP-1 gene pBK-CMV derivative carrying a pMB3011 gDNA fragment from P. aeruginosa This study NRZ-00156 including blaIMP-31 pBK-CMV derivative carrying a pMB3013 gDNA fragment from P. aeruginosa Hoffmann (2013) NRZ-03096 including blaKHM-2 pBK-CMV derivative carrying the pMB3014 Hoffmann (2013) blaKHM-2 gene Wildtype plasmid from C. freundii pMB3018 This study NRZ-02127 pBK-CMV derivative carrying the Lange (2014) pMB3026 blaOXA-10 gene pEX-A2 derivative carrying the pEX-A2-KHM-1 This study synthesized blaKHM-1 gene pBK-CMV derivative carrying the pMB3037 This study blaKHM-1 gene Material and Methods 29
2.2.3 Oligonucleotides All Oligonucleotides used in this study are listed in Table 2.3.
Table 2.3: Oligonucleotides used in this study. Recognition sites for restriction endonucleases are underlined.
Oligonucleotide Sequence (5´to 3´) Purpose Reference
Sequencing of blaIMP-31 ACG CAG CAG GGC AGT IMP-X_seq_fw from gDNA of P. aeruginosa This study CGC C NRZ-00156
Sequencing of blaIMP-31 GTT GGG GCA GTC CCG IMP-X_seq_rev from gDNA of P. aeruginosa This study CTT GG NRZ-00156
Sequencing of blaIMP-31 AAT ACT GCC TTT GAT IMP-X_seq_up from gDNA of P. aeruginosa This study TTT AT NRZ-00156
Sequencing of blaIMP-31 GAA AAC TCA TTT AGT IMP-X_seq_dwn from gDNA of P. aeruginosa This study GGC GT NRZ-00156
Sequencing of blaIMP-31 ACG CAG CAG GGC AGT IMP-X_seq_fw from gDNA of P. aeruginosa This study CGC C NRZ-00156
Sequencing of blaIMP-31 GTT GGG GCA GTC CCG IMP-X_seq_rev from gDNA of P. aeruginosa This study CTT GG NRZ-00156
Sequencing of blaIMP-31 AAT ACT GCC TTT GAT IMP-X_seq_up from gDNA of P. aeruginosa This study TTT AT NRZ-00156
Sequencing of the blaIMP-31 GAG CTT CTT AAA AAG GW-IMP-31-GSP1 genetic context from This study AAC GGT AAT GCG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GCC GAA AAC TCA TTT GW-IMP-31-GSP2 genetic context from This study AGT GGC GTT AGC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CTC AGC CCC TTA GCT CTG GW-IMP-31-GSP3 genetic context from This study CGT TAG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CTT GGG AGC AGG CTG GW-IMP-31-GSP2-5 genetic context from This study TTA AGG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GGC AAC CAG AAT ATC GW-IMP-31-GSP4 genetic context from This study AGT GGT G P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GTA CCT CGC TGT TGG CCA GW-IMP-31-GSP5 genetic context from This study GGT CGA AAC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CCC GTA TTT CAA CAA GW-IMP-31-GSP5-1 genetic context from This study ATC GCC AG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CAA AGT ACA GCA TCG GW-IMP-31-GSP6 genetic context from This study TGA CCA AC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CCG AGC AAC TTG CGA GW-IMP-31-GSP7 genetic context from This study GCG ATC CG P. aeruginosa NRZ-00156 Material and Methods 30
Oligonucleotide Sequence (5´to 3´) Purpose Reference
Sequencing of the blaIMP-31 GAG CTC TGG TTG AGT GW-IMP-31-GSP7-1 genetic context from This study TGC TGT TC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GCC GTG TAC ATG GTT GW-IMP-31-GSP8 genetic context from This study CAA ACA CGC C P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GCG AGC GAT CCG ATG GW-IMP-31-GSP8-1 genetic context from This study CTA CGA GAA AG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CAG ATG GTC CAG CCG GW-IMP-31-GSP9 genetic context from This study TGT ACA TG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GGT GAA TGC GGG AAA GW-IMP-31-GSP9-1 genetic context from This study CGT TAA GTG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GAG GCT TTT GAG GAC GW-IMP-31-GSP10 genetic context from This study GCT GAG AAC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GCC CAT ATG GCA CGA GW-IMP-31-GSP11 genetic context from This study TCG TTT CG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GCC GCA GAC GCC TCA TAT GW-IMP-31GSP11-1 genetic context from This study GTA TAT C P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CTG CTC AAG AAA TTC GW-IMP-31GSP11-2 genetic context from This study TAC AAG AGC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GCC GTG TAC ATG GTT GW-IMP-31GSP12 genetic context from This study CAA ACA CG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CAT TGA CGC GGT ATT GW-IMP-31-GSP13 genetic context from This study TGG ACC AG P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CTG GAA ATG TAT CTC GW-IMP-31GSP14 genetic context from This study AAC CAG C P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CAA GTG AGG GCA TCA GW-IMP-31GSP15 genetic context from This study TTG GTG GC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GAT CTG CGC CAC CTG ATC GW-IMP-31-AP3 genetic context from This study AAC ACT G P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GCC TTG CGC ACC TTT ACG GW-IMP-31-AP3-1 genetic context from This study AGG ATC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 CGA ATT GTT AGA CCG GW-IMP-31-AP4 genetic context from This study CGC TTA GAA G P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GAT ACT TCG TCG AGG GW-IMP-31-AP5 genetic context from This study GCG ACT GTC P. aeruginosa NRZ-00156
Sequencing of the blaIMP-31 GTA AAG CTG CTC TCC CTC GW-IMP-31-AP6 genetic context from This study GGT TC P. aeruginosa NRZ-00156 Material and Methods 31
Oligonucleotide Sequence (5´to 3´) Purpose Reference
Sequencing of the blaIMP-31 CTC GAT GGA AGG GTT GW-IMP-31-AP7 genetic context from This study AGG CAT C P. aeruginosa NRZ-00156 CGA CGT TGT AAA ACG Sequencing of pBK-CMV M13_uni New England Biolabs ACG GCC AGT based plasmids Sequencing of pBK-CMV M13_rev CAG GAA ACA GCT ATG AC New England Biolabs based plasmids GGC CGC CCC TCA TGT CAA OXA-X_seq.fw Sequencing of pMB3002 Meining (2012) AC TAG AAT GGC TCT CCC TTT pMB3002_seq_fw1 Sequencing of pMB3002 Meining (2012) TC ATG TCC TCT GGT AAA pMB3002_seq_fw.2 Sequencing of pMB3002 Meining (2012) CGG GT CTC ACT GCT TCT GCG CTG pMB3002_seq_fw3 Sequencing of pMB3002 Meining (2012) TT ATC TTC AAA GTC CGG pMB3002_seq_fw4 Sequencing of pMB3002 Meining (2012) CAT CG TTT ACG AAG TTT CTC pMB3002_seq_fw5 Sequencing of pMB3002 Meining (2012) ACC GCC ACG GCT TCG GCA GAG pMB3002_seq_rev.1 Sequencing of pMB3002 Meining (2012) AAC TC GCC ACT CAT AGA GCA pMB3002_seq_rev.2 Sequencing of pMB3002 Meining (2012) TCG CA GGG TCA AGG ATC TGG pMB3002_seq_rev.3 Sequencing of pMB3002 Meining (2012) ATT TC GTC GGC TTC TGA CGT pMB3002_seq_rev.4 Sequencing of pMB3002 Meining (2012) TCA GT CCA TTA ATG TTC CGC pMB3002_seq_rev5 Sequencing of pMB3002 Meining (2012) AAA TC ACG GCT TCG GCA GAG OXA233_Int_rev Sequencing of pMB3002 Meining (2012) AAC TC AGG CGG CAC CTG AAT OXA233_Int_fw Sequencing of pMB3002 Meining (2012) ATC TAG T TGT TCA ATG ATC CCG Oxa233_Int2_fw2 Sequencing of pMB3002 This study AGG TC TCA TAG AGC ATC GCA Oxa233_Int2_rev2 Sequencing of pMB3002 This study AGG TC CAT TGC AAT GCT GAA Oxa233_Int1_fw2 Sequencing of pMB3002 This study TGG AG CGA GGT CAC CAA GAT Oxa233_Int1_fw3 Sequencing of pMB3002 This study CCA AA TTC GTG CCT TCA TCC GTT Oxa233_Int1_rev3 Sequencing of pMB3002 This study TC CGA CAG GTG CCG GCA pMB3013_seq_fw1 Sequencing of pMB3013 Hoffmann (2013) CAC GCG ATG CAG AAC AAA CTA ATG pMB3013_seq_rv1 Sequencing of pMB3013 Hoffmann (2013) AAT TGC GAT GTG CGC AAC GCA pMB3013_seq_fw2 Sequencing of pMB3013 Hoffmann (2013) GAA C CCT GTC TTT GAC AAG pMB3013_seq_rv2 Sequencing of pMB3013 Hoffmann (2013) CAG ACC CAC GGC GTC TTG AGC pMB3013_seq_rv3 Sequencing of pMB3013 Hoffmann (2013) TGA TAC Material and Methods 32
Oligonucleotide Sequence (5´to 3´) Purpose Reference Verification of gene GGC TGA GGT TCG ACG 3014-isx-khm_fw arrangement of the genetic This study CTA ATC AG context of blaKHM-2 Verification of gene GGG TTT TAC AAA ACA 3014-isx-khm_rev arrangement of the genetic This study GCC ACC G context of blaKHM-2 Verification of gene GTA CCG GGT CAT GGA 3014-KHM-aac3_fw arrangement of the genetic This study ACA ATG G context of blaKHM-2 Verification of gene CCG TAT TGC AGA GGA 3014-KHM-aac3_rev arrangement of the genetic This study TGG TTC TC context of blaKHM-2 Verification of gene CTC AGG AAC TGA CTG 3014-aac3-insE_fw arrangement of the genetic This study CCT TCG C context of blaKHM-2 Verification of gene GTA CGG AAA ACT CAG 3014-aac3-insE_rev arrangement of the genetic This study CAC CCA TTG context of blaKHM-2 GGA ATA GAG TGG CTT -lactamase Rossolini & Docquier IMP-DIA_fw AAT TCT C genes (2007) Detection of β GTG ATG CGT CYC CAA -lactamase Rossolini & Docquier IMP-DIA_rev YTT CAC T genes (2007) Detection of β GTY CTT TCG AGT ACG -lactamase OXA-10A Nordmann & Naas (2010) GCA TTA genes Detection of β ATT TTC TTA GCG GCA -lactamase OXA-10B Nordmann & Naas (2010) ACT TAC genes Detection of β TTG GTG GCA TCG ATT -lactamase OXA-48A Poirel et al. (2004b) ATC GG genes Detection of β GAG CAC TTC TTT TGT -lactamase OXA-48B Poirel et al. (2004b) GAT GGC genes Detection of β -lactamase GAA GGY GTT TAT GTT IMP-A genes and blaIMP-31 DIG Pitout et al. (2005) CAT AC probeDetection synthesis of β -lactamase GTA mgT TTC AAG AGT IMP-B genes and blaIMP-31 DIG Pitout et al. (2005) GAT GC probeDetection synthesis of β GTT TGG TCG CAT ATC -lactamase VIM_2004A Pitout et al. (2005) GCA AC genes Detection of β AAT GCG CAG CAC CAG -lactamase VIM_2004B Pitout et al. (2005) GAT AG genes Detection of β AGT GGT GAG TAT CCG -lactamase VIM-F Juan et al. (2008) ACA G genes Detection of β ATG AAA GTG CGT GGA -lactamase VIM-R Juan et al. (2008) GAC genes Detection of β CCT ACA ATC TAA CGG -lactamase SPM-1F Castanheira et al. (2004) CGA CC genes Detection of β TCG CCG TGT CCA GGT -lactamase SPM-1R Castanheira et al. (2004) ATA AC genes Detection of β AGA ACC TTG ACC GAA -lactamase GIM-1F Castanheira et al. (2004) CGC AG genes Detection of β ACT CAT GAC TCC TCA CGA -lactamase GIM-1R Castanheira et al. (2004) GG genes Detection of β Material and Methods 33
Oligonucleotide Sequence (5´to 3´) Purpose Reference TAC AAG GGA TTC GGC -lactamase SIM1-F Lee et al. (2005) ATC G genes Detection of β TAA TGG CCT GTT CCC -lactamase SIM1-R Lee et al. (2005) ATG TG genes Detection of β CAA TAT TAT GCA CCC -lactamase NDM-1_a_fw Kaase (unpublished) GGT CG genes Detection of β CCT TGC TGT CCT TGA TCA -lactamase NDM-1_a_rev Kaase (unpublished) GG genes Detection of β GTA CCT GAG CTA AGA -lactamase DIM-1_a_fw Kaase (unpublished) ATC GAG genes Detection of β CGG CTG GAT TGA TTT -lactamase DIM-1_a_rev_neu Kaase (unpublished) GTT AGA G genes Detection of β GAA ACG TCG CTT CAC CCT -lactamase AIM-1_a_fw Kaase (unpublished) G genes Detection of β ACC AGG ATG TCG CAG Detection of -lactamase AIM-1_a_rev Kaase (unpublished) TCG AG genes β GCT CTT GTT ATA TCG -lactamase KHM-1_a_fw Kaase (unpublished) TTT GGT C genes Detection of β CAT TGT TGC ATT GCT -lactamase KHM-1_a_rev Kaase (unpublished) ATA ACG G genes Detection of β CCT CAA CTG GAT CAA -lactamase NDM-1_b_fw Kaase (unpublished) GCA GG genes Detection of β GAC AAC GCA TTG GCA -lactamase NDM-1_b_rev Kaase (unpublished) TAA GTC genes Detection of β GAA GCA CAT GGA AAA -lactamase FIM-1_F Pollini et al. (2013) CTG GG genes Detection of β GAT GGG CGA ATG AGA -lactamase FIM-1_R Pollini et al. (2013) CAG C genes Detection of β ATA GCC ATC CTT GTT -lactamase IMI-A Aubron et al. (2005) TAG CTC genes Detection of β TCT GCG ATT ACT TTA TCC -lactamase IMI-B Aubron et al. (2005) TC genes Detection of β GTT TTG CAA TGT GCT -lactamase Weldhagen & Prinsloo GES-C CAA CG genes (2004) Detection of β TGC CAT AGC AAT AGG -lactamase Weldhagen & Prinsloo GES-D CGT AG genes (2004) Detection of β -lactamase KPC_5 TGT CAC TGT ATC GCC GTC Yigit et al. (2001) genes Detection of β CTC AGT GCT CTA CAG -lactamase KPC_10 Yigit et al. (2001) AAA ACC genes Detection of β AAC AAG GAA TAT CGT -lactamase KPC_fw Pasteran et al. (2008) TGA TG genes Detection of β AGA TGA TTT TCA GAG -lactamase KPC_rev Pasteran et al. (2008) CCT TA genes Detection of β CTG TAT CGC CGT CTA GTT -lactamase KPC_a_fw Kaase (unpublished) CTG genes Detection of β GTC GTG TTT CCC TTT AGC -lactamase KPC_a_rev Kaase (unpublished) CA genes Detection of β AAT ATC TGA CAA CAG -lactamase KPC_b_fw Kaase (unpublished) GCA TGA CGG genes Detection of β GTT GAC GCC CAA TCC CTC -lactamase KPC_b_rev Kaase (unpublished) GA genes Detection of β Material and Methods 34
Oligonucleotide Sequence (5´to 3´) Purpose Reference CCG CCG CCA ATT TGT TGC Detection of -lactamase KPC_c_fw Kaase (unpublished) TG genes β TTA CTG CCC GTT GAC GCC -lactamase KPC_c_rev Kaase (unpublished) CA genes Detection of β Sequencing of integrons or AAA TCC ATT CCC ACC OXA-10_end.fw genes correlated with This study AAA ATC A integrons Sequencing of integrons or ATG TCT AAC TTT GTT aadA6_start_rev genes correlated with This study TTA GGG CGA C integrons Sequencing of integrons or GAG CGG AAT GTA GTG aadA6_end_fw genes correlated with This study CTT ACC TT integrons Sequencing of integrons or integron_5CS GGC ATC CAA GCA GCA AG genes correlated with Levesque et al. (1995) integrons Sequencing of integrons or integron_3CS AAG CAG ACT TGA CCT GA genes correlated with Levesque et al. (1995) integrons Sequencing of integrons or CTC TCA CTA GTG AGG INT-F genes correlated with Juan et al. (2008) GGC integrons Sequencing of integrons or ATG AAA ACC GCC ACT INT-R genes correlated with Juan et al. (2008) GCG integrons Sequencing of integrons or integron_5CS GGC ATC CAA GCA GCA AG genes correlated with Falcone et al. (2009) integrons Sequencing of integrons or CTC TCA AGA TTT TAA integron_3CS genes correlated with Falcone et al. (2009) TGC GGA TG integrons Sequencing of integrons or GCC AAC TAT TGC GAT qacE_delta1 genes correlated with Schneider et al. (2008) AAC integrons Sequencing of integrons or GAA AGG CTG GCT TTT qacE-F genes correlated with Juan et al. (2008) TCT TG integrons Sequencing of integrons or ATT ATG ACG ACG CCG qacE-R genes correlated with Juan et al. (2008) AGT C integrons Sequencing of integrons or GCC TGT TCG GTT CGT int1_fw genes correlated with Toleman et al. (2005) AAG CT integrons Sequencing of integrons or CGG ATG TTG CGA TTA QacR_rev genes correlated with Toleman et al. (2005) CTT CG integrons Sequencing of integrons or GGA GCA GCA ACG ATG aadA6_fw genes correlated with This study TTA CG integrons Sequencing of integrons or TTG CTG CGC TGT ACC aadA6_rev genes correlated with This study AAA TG integrons Material and Methods 35
Oligonucleotide Sequence (5´to 3´) Purpose Reference Sequencing of integrons or CCG ACT TCA GCT TTT sul1_class1_rev genes correlated with This study GAA GGT TC integrons Sequencing of integrons or CAA TTA TGA GCC CCA qacE_class1_rev genes correlated with This study TAC CTA CAA AG integrons ACC TGG TGT ACG CCT CGC MLS-typing of acsA-F_ampl_curran Curran et al. (2004) TGA C P. aeruginosa NRZ-00156 GAC ATA GAT GCC CTG CCC MLS-typing of acsA-R_ampl_curran Curran et al. (2004) CTT GAT P. aeruginosa NRZ-00156 GCC ACA CCT ACA TCG TCT MLS-typing of acsA-F_seq_curran Curran et al. (2004) AT P. aeruginosa NRZ-00156 AGG TTG CCG AGG TTG MLS-typing of acsA-R_seq_curran Curran et al. (2004) TCC AC P. aeruginosa NRZ-00156 TGG GGC TAT GAC TGG MLS-typing of aroE-F_ampl_curran Curran et al. (2004) AAA CC P. aeruginosa NRZ-00156 TAA CCC GGT TTT GTG MLS-typing of aroE-R_ampl_curran Curran et al. (2004) ATT CCT ACA P. aeruginosa NRZ-00156 ATG TCA CCG TGC CGT TCA MLS-typing of aroE-F_seq_curran Curran et al. (2004) AG P. aeruginosa NRZ-00156 TGA AGG CAG TCG GTT MLS-typing of aroE-R_seq_curran Curran et al. (2004) CCT TG P. aeruginosa NRZ-00156 CGG CCT CGA CGT GTG MLS-typing of guaA-F_ampl_curran Curran et al. (2004) GAT GA P. aeruginosa NRZ-00156 GAA CGC CTG GCT GGT MLS-typing of guaA-R_ampl_curran Curran et al. (2004) CTT GTG GTA P. aeruginosa NRZ-00156 AGG TCG GTT CCT CCA MLS-typing of guaA-F_seq_curran Curran et al. (2004) AGG TC P. aeruginosa NRZ-00156 GAC GTT GTG GTG CGA MLS-typing of guaA-R_seq_curran Curran et al. (2004) CTT GA P. aeruginosa NRZ-00156 CCA GAT CGC CGC CGG TGA MLS-typing of mutL-F_ampl_curran Curran et al. (2004) GGT G P. aeruginosa NRZ-00156 CAG GGT GCC ATA GAG MLS-typing of mutL-R_ampl_curran Curran et al. (2004) GAA GTC P. aeruginosa NRZ-00156 AGA AGA CCG AGT TCG MLS-typing of mutL-F_seq_curran Curran et al. (2004) ACC AT P. aeruginosa NRZ-00156 GGT GCC ATA GAG GAA MLS-typing of mutL-R_seq_curran Curran et al. (2004) GTC AT P. aeruginosa NRZ-00156 MLS-typing of nuoD-F_ampl_curran ACC GCC ACC CGT ACT G Curran et al. (2004) P. aeruginosa NRZ-00156 MLS-typing of nuoD-R_ampl_curran TCT CGC CCA TCT TGA CCA Curran et al. (2004) P. aeruginosa NRZ-00156 ACG GCG AGA ACG AGG MLS-typing of nuoD-F_seq_curran Curran et al. (2004) ACT AC P. aeruginosa NRZ-00156 TGG CGG TCG GTG AAG MLS-typing of nuoD-R_seq_curran Curran et al. (2004) GTG AA P. aeruginosa NRZ-00156 GGT CGC TCG GTC AAG MLS-typing of ppsA-F_ampl_curran Curran et al. (2004) GTA GTG G P. aeruginosa NRZ-00156 GGG TTC TCT TCT TCC GGC MLS-typing of ppsA-R_ampl_curran Curran et al. (2004) TCG TAG P. aeruginosa NRZ-00156 GGT GAC GAC GGC AAG MLS-typing of ppsA-F_seq_curran Curran et al. (2004) CTG TA P. aeruginosa NRZ-00156 Material and Methods 36
Oligonucleotide Sequence (5´to 3´) Purpose Reference GTA TCG CCT TCG GCA MLS-typing of ppsA-R_seq_curran Curran et al. (2004) CAG GA P. aeruginosa NRZ-00156 GCG GCC CAG GGT CGT MLS-typing of trpE-F_ampl_curran Curran et al. (2004) GAG P. aeruginosa NRZ-00156 CCC GGC GCT TGT TGA MLS-typing of trpE-R_ampl_curran Curran et al. (2004) TGG TT P. aeruginosa NRZ-00156 TTC AAC TTC GGC GAC TTC MLS-typing of trpE-F_seq_curran Curran et al. (2004) CA P. aeruginosa NRZ-00156 GGT GTC CAT GTT GCC MLS-typing of trpE-R_seq_curran Curran et al. (2004) GTT CC P. aeruginosa NRZ-00156
AAA AGG ATC CGC CCT Cloning of blaIMP-31 into IMP-31_Bam_fw This study AAA ACA AAG TTA GAA pBK-CMV
TTT TAA GCT TTT ATT Cloning of blaIMP-31 into IMP-31_Hind_rev This study TGG GGC TGT GAT pBK-CMV
AAA AGG ATC CGT CGC Cloning of blaIMP-1 into IMP-1_BamHI_fw This study CCT AAA ACA AAG TTA G pBK-CMV
TTT TCT CGA GTT AGT Cloning of blaIMP-1 into IMP-1_XhoI_rev This study TGC TTG GTT TTG ATG pBK-CMV
AAA AGG ATC CTT AGC Cloning of blaOXA-233 into OXA-233+20up_F Meining (2012) CAC CAA GAA GGT GCC pBK-CMV
TTT TAA GCT TTT AGC Cloning of blaOXA-233 into OXA-X_HindIII_rev Meining (2012) CAC CAA TGA TGC CC pBK-CMV AAA AGA GCT CAC GGC Cloning of blaOXA-10 into OXA10Klon_fwdSac TTA ATT CTG GCG TTA Lange (2014) pBK-CMV GCC ACC AAG AAG GTG CC
AAA AGG TAC CTT AGC Cloning of blaOXA-10 into OXA10Klon_revKpn Lange (2014) CAC CAA TGA TGC CCT C pBK-CMV
AAA AGG ATC CAA TTT Cloning of blaKHM-2 into KHM-2-BamHI-fw Hoffmann (2013) AAT CGC ACG AAT AG pBK-CMV
TTT TCT CGA GTT ATT Cloning of blaKHM-2 into KHM-2-XhoI-rev Hoffmann (2013) TCT TCT TTG CAA CC pBK-CMV AAA AGG ATC CAT TTC Cloning of blaKHM-1 into KHM-1_Bam_fw TCA ATA AAA ATA TAG This study pBK-CMV AAG G
TTT TAA GCT TTC ACT Cloning of blaKHM-1 into KHM-1_Hind_rev This study TTT TAG CTG CAA GC pBK-CMV CAG CAG CCG CGG TAA 16-rRNA DIG probe 536F_fournier Fournier et al. (2010b) TAC synthesis ACG GCT ACC TTG TTA 16-rRNA DIG probe RP2_fournier Fournier et al. (2010b) CGA CTT synthesis
GTT TGG TTT TTG TGG blaKHM-2 DIG probe KHM-2_DIG_fw2 This study ATG GT synthesis
CGA TTG ATA AGT TTT blaKHM-2 DIG probe KHM-2_DIG_fw2 This study TCT GC synthesis
Material and Methods 37
2.3 Methods
2.3.1 Microbiological methods
2.3.1.1 Growth and storage of bacterial cells Bacterial cells were grown at 37 °C on LB, MacConkey, Mueller-Hinton and Columbia blood agar plates or in liquid LB medium supplemented with appropriate antibiotics, if necessary. Agar plates containing bacterial colonies were stored at 4 °C. For longterm storage, 800 µl of 88 % glycerol were added to 800 µl of an overnight culture and the mixture was frozen in liquid nitrogen. The cultures were stored at -80 °C.
LB medium 1 % (w/v) NaCl (AppliChem) 1 % (w/v) Tryptone 0.5 % (w/v) Yeast extract
LB agar 1.5 % (w/v) Agar (AppliChem) in LB medium
MacConkey agar 1.7 % (w/v) Peptone from gelatin (Merck) 0.15 % (w/v) Peptone from casein 0.15 % (w/v) Peptone from meat 0.5 % (w/v) NaCl 1 % (w/v) Lactose 0.15 % (w/v) Bile salt mixture 0.003 % (w/v) Neutralred 0.0001 % (w/v) Crystalviolett 1.35 % Agar
Columbia blood agar 2.3 % (w/v) Peptone (Thermo Scientific Oxoid) 0.1 % (w/v) Starch 1 % (w/v) NaCl 1 % (w/v) Agar 4 % (v/v) Defibrinated sheep blood
Material and Methods 38
2.3.1.2 Determination of bacterial growth in fluid cultures Bacterial growth was determined by measuring the optical density (OD) of a culture at a wavelength of 600 nm. A OD600 of 1 corresponds to 1-5 x 108 cells per ml. Turbidity of bacterial suspensions in 0.8 % NaCl was optically measured using a VITEK Densichek (bioMérieux) in relation to McFarland standards.
2.3.1.3 Preparation of chemically competent E. coli cells 100 ml of LB medium were supplemented with 2 ml Mg2+ solution and inoculated with 2 ml of an overnight E. coli culture. The cells were grown to an OD600 of 0.5 at 37 °C. The cells were harvested by centrifugation (4,000 × g, 4 °C, 10 min) and the pellet was resuspended in 50 ml TMF buffer, previously cooled to 4 °C. After 1 h incubation on ice the cells were harvested again (3,000 × g, 4 °C, 10 min) and the pellet was resuspended in 10 ml of cold TMF buffer and supplemented with 3 ml of 88 % glycerol. The cells were aliquoted in reaction tubes to 250 µl each and stored at -80 °C.
TMF buffer 40 mM MnCl2 50 mM RbCl
Mg2+ solution 500 mM MgCl2
500 mM MgSO4
2.3.1.4 Transformation of competent E. coli cells A 250 µl aliquot of competent E. coli cells was thawed on ice. After addition of 1-3 µl of purified plasmid DNA or 20 µl of a ligation mixture the cells were incubated on ice for 30 min. Transformation was performed by heat shocking at 42 °C for 2 min. After heat shock 700 µl of LB medium were added, followed by 1 h incubation at 37 °C at 300 rpm in an incubation shaker. The cells were harvested by centrifugation (16,000 × g, 1 min), the supernatant was discarded except for approx. 100 µl and the cell pellet was resuspended in the remaining supernatant. The cells were plated on LB agar plates containing appropriate antibiotics and were incubated overnight at 37 °C.
2.3.1.5 Conjugation experiments For conjugation experiments, bacterial isolates and a recipient strain were mixed and inoculated as a spot of around 1 cm onto Columbia blood agar plates. As recipient strains either E. coli C600 (rifampicin resistant) or E. coli J53 (sodium azide resistant) were used. After 18 h of incubation transconjugants were selected on LB agar containing 100 mg/l ampicillin and selective antibiotic (100 mg/l rifampicin or 200 mg/l sodium azide). Material and Methods 39
2.3.2 Phenotypic methods for antibiotic resistance analysis
2.3.2.1 Disk diffusion antibiotic susceptibility test Resistance to antibiotics was analyzed using paper disks containing single antibiotics or -lactamase inhibitors (Thermo Scientific Oxoid). Bacteria from combinationsagar plates or of liquid antibiotics cultures and were β suspended in 0.8 % NaCl until a McFarland of 0.5-0.8 was reached. MH2 agar plates (bioMérieux) were evenly inoculated with bacteria using a sterile cotton tip. Antibiotic disks were placed on the plate with at least 1 cm distance from each other or the agar edge. The plates were incubated overnight at 37 °C and resistance to antibiotics was evaluated by measuring the inhibition zone diameters. The results were interpreted following the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).
2.3.2.2 EDTA-Combined disk test (EDTA-CDT) For phenotypic analysis of MBL production, the analyzed strain was plated on MH2 agar plates as described in 2.3.2.1. Antibiotik disks containing carbapenems were placed on the plate in duplicates and 10 µl of 0.5 M EDTA were pipetted on the disk. The plates were incubated overnight at 37 °C and the inhibition zone diameters were measured. An increased inhibition zone of the carbapenem/EDTA disk compared to the one without EDTA indicated an MBL production.
2.3.2.3 Modified Hodge Test To analyze if a bacterial strain was producing a carbapenemase, MH2 agar plates were inoculated with E. coli ATCC 25922 as described in 2.3.2.1. Disks containing imipenem, meropenem and ertapenem were placed on the inoculated agar and colony material from the test strain was streaked in a straight line from the edge of one disk to another disk. The plates were incubated overnight at 37 °C and the plate was examinend for clover leaf-like indentation of E.coli ATCC 25922 growth along of the the test strain streak within the disk diffusion zone. A growth of E.coli ATCC 25922 into the zone was interpreted as positive and no growth as negative.
2.3.2.4 Determination of minimal inhibitory concentration (MIC) Determination of MICs was performed using the Etest gradient test strips (bioMérieux). MH2 agar plates were inoculated with bacteria as described in 2.3.2.1. The gradient strips were placed onto the agar plate with sterile tweezers. The plates were incubated overnight at 37 °C. The MIC was determined by reading the scale of the gradient strip at the position were bacterial growth reached the strip. MIC values of clinical isolates were interpreted following the EUCAST guidelines (http://www.eucast.org/clinical_breakpoints/; 15 March 2015, date last accessed) (EUCAST, 2015) Material and Methods 40
2.3.3 Molecular biology methods
2.3.3.1 Preparation of genomic DNA from Gram-negative bacteria Genomic DNA from Gram-negative bacteria was extracted using the NucleoSpin Tissue kit (Macherey-Nagel) following the manufacturer’s instructions.
2.3.3.2 Preparation of plasmid DNA from E. coli Plasmid DNA from E. coli was extracted using the NucleoSpin Plasmid kit (Macherey-Nagel) following the manufacturer’s instructions. For plasmids larger than 10 kb, the NucleoBond® PC 100 kit was used following the manufacturer’s instructions.
2.3.3.3 Purification of PCR-Products and DNA fragments from agarose gels PCR products and DNA fragments from agarose gels were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) following the manufacturer’s instructions.
2.3.3.4 Quick preparation of plasmid DNA from E. coli cells For quick extraction of plasmid DNA from E. coli, the procedure described by Holmes & Quigley (1981) was used with several modifications. Cells were grown in 4 ml of LB media overnight and harvested by centrifugation (16,000 × g, 1 min). The pellet was resuspended in 50 µl boiling- prep buffer and incubated for 3 min at room temperature, followed by incubation at 95 °C for 1 min. The samples were cooled on ice for 5 min and centrifuged (16,000 × g, 4 °C, 10 min). The supernatant contained the plasmid DNA.
Boiling-prep buffer 10 mM Tris/HCL, pH 8.0 (Storage at -20 °C) 1 mM EDTA 15 % (w/v) Sucrose 2 mg/ml Lysozym 0.2 mg/ml RNAseA 0.1 mg/ml BSA
2.3.3.5 Agarose gel electrophoresis of DNA fragments To determine the size of DNA fragments and for preparative purifaction of PCR products, DNA fragments were separated by agarose gel electrophoresis using gels containing 0.8 % (w/v) agarose in TBE buffer. The samples were mixed with loading buffer and applied to a gel. Separation of DNA fragments was performed at a constant voltage of 50-150 V. After electrophoresis, the gel was incubated in 0.05 % ethidium bromide for 10-30 min. After a washing step in A. dest, the DNA bands were visualized under UV illumination using the Gel Doc Material and Methods 41
XR+ system (Bio-Rad). The GeneRulerTM DNA Ladder Mix (Thermo Scientific) served as a size marker.
TBE-buffer 89 mM Tris 89 mM Boronic acid 2 mM EDTA pH 7.8
Loading buffer 0.5 % (w/v) Bromphenol blue 43 % (v/v) Glycerol 100 mM EDTA
2.3.3.6 Digestion of bacterial DNA with restriction endonucleases Restriction of bacterial DNA was performed using FastDigest® restriction endonucleases and the FastDigest buffer system (Thermo Scientific). The reactions contained 1-3 µl of appropriate endonucleases and up to 16 µl of purified DNA and were carried out at 37 °C for 2-30 min in a total volume of 20 µl. Restriction of vector DNA for ligation purposes additionally contained 1 µl of FastAP alkaline phosphatase to prevent religation by dephosphorylation of the restricted vector ends. For of ligation reactions, the enzymes were inactivated at 65-80 °C for 5-10 min after restriction.
2.3.3.7 Ligation of DNA fragments Ligation of DNA fragments with T4 DNA ligase was performed by mixing 1 µl of restricted and dephosphorylated target vector DNA with 0.5-6 µl of the desired restricted DNA insert. The DNA was incubated with 1 µl of T4 DNA ligase in 1 x T4 DNA ligase buffer (Thermo Scientific) in a total volume of 20 µl for 1 h or overnight at room temperature.
2.3.3.8 Polymerase chain reaction (PCR) for amplification of DNA fragments Amplifikation of DNA fragments up to 2 kb was performed as described by Saiki et al. (1988) using Taq, Pfu and Pwo DNA polymerases in their appropriate buffer. For amplification of larger DNA fragments (up to 8 kb), the FastStart High Fidelity kit (Roche) or the Expand Long Range PCR System (Roche) were used following the manufacturer’s instructions and the supplied buffers.
2.3.3.9 Genome walking To amplify and sequence larger parts of unknown genomic DNA starting from a known gene sequence, the Universal GenomeWalker 2.0 kit (Clontech) was used following the Material and Methods 42
manufacturer’s instructions. Total DNA of bacteria was digested with various restriction endonucleases to obtain uncloned DNA libraries. The DNA fragments were ligated with adaptor nucleotides with known sequences. The fragment-adaptor nucleotides were amplified by PCR with an adaptor-specific and a gene-specific oligonuncleotide and the products were sequenced (2.3.3.12).
2.3.3.10 DNA synthesis Synthesis of bacterial DNA was performed by Eurofins Genomics and provided subcloned into the pEX-A2 vector.
2.3.3.11 Determination of DNA and protein concentration in aqueous solutions DNA concentrations and quality were determined photometrically using the Eppendorf BioSpectrometer with the Eppendorf µCuvette G1.0 by measuring the absorbance of a sample at 260 and 280 nm. Protein concentrations were either determined using the modified Bradford assay Roti-Nanoquant (Roth) following the manufacturer’s instructions or using the Qubit 2.0 fluorometer with the Qubit Protein Assay Kit (Life Technologies).
2.3.3.12 Sequencing of plasmid and genomic DNA Sequencing of bacterial DNA was performed in publication quality with Sanger-sequencing by third-party companies (Seqlab, GATC) following the companies DNA sample requirements.
2.3.3.13 Sequencing of plasmid DNA by 454-pyrosequencing Large wildtype plasmids were sequenced using the 454 next generation sequencing technique and the GS Junior system (Roche) at the Department of Virology (RUB) following the manufacturer’s manuals. 300 ng of purified plasmid DNA were fragmented by nebulization and damaged ends were repaired by T4 Ligase and Taq Polymerase. An adaptor molecule was ligated with the DNA fragments and the fragments were bound to magnetic beads. Small fragments were removed by washing and pelleting steps using the magnetic beads within a magnetic tube holder. The library quality was analyzed by gel electrophoresis after separating the DNA fragments from the magnetic beads by addition of TE buffer. The library was quantified fluorometrically and the library sample concentration was calculated on the basis of a standard curve. The DNA library was bound to capture beads and amplified by random amplified PCR in a 96-well plate. After amplification, the library was recovered from the plate and washed with ethanol and isopropanol for removal of PCR reagents. The library was transferred to Enrichment beads by several washing steps and the sequencing oligonucleotides were annealed. The library was adjusted to a total amount of approx. 500,000 enriched beads, determined using the GS Junior Bead Counter. 454 sequencing was performed by loading the bead-bound DNA library on Material and Methods 43
a GS Junior Titanium Pico TiterPlate, preloaded with the enzymes required for the sequencing reaction. The sequence of the DNA fragments was determined by single nucleotide addition. If a nucleotide is ligated to the single stranded DNA, the released pyrophosphate is converted into ATP by an ATP sulfurylase and used by a luciferase to convert luciferin to oxyluciferin which leads to light emission. The light emission is detected by a bioluminescence camera and the DNA is sequenced. After sequencing, the DNA sequence fragments were assembled using the GS Junior Sequencer software.
2.3.3.14 Shotgun-cloning approach for the identification of bacterial resistance genes -lactam resistance of Gram-
Inneg orderative toclinical determine isolates the, moleculargenomic DNA mechanism of the of isolates increased was β digested with restriction endonucleases whose specific recognition sites statistically occur with high frequency in Gram- negative bacterial DNA. Namely, BamHI (GGATCC), HindIII (AAGCTT), EcoRI (GAATCC), XhoI (CTCGAG), MboI and Sau3AI (both GATC) were used. The aim was to receive relatively small DNA fragments with sizes from 0.5-6 kb. For MboI, digestion was performed for only 2-5 min, as the MboI recognition site (GATC) is very frequent in bacterial DNA and a longer incubation would lead to DNA fragments too small for efficient cloning. After restriction, the DNA fragments were ligated (2.3.3.7) with the pBK-CMV vector which was previously digested with the same restriction enzyme (for MboI: BamHI) and the ligation preparation was transformed into E. coli TOP10 (2.3.1.4). The cells were plated on LB agar plates containing 50-100 mg/l ampicillin and 50 mg/l kanamycin to select for cells that received complete genes that confer higher levels of - lactam resistance. The plates were incubated overnight at 37 °C. In case of bacterial growth,β plasmid DNA was extracted from the cells (2.3.3.2) and the insert of the pBK-CMV vector was sequenced using appropriate oligonucleotides (Table 2.3 & 2.3.3.12).
2.3.3.15 Multilocus sequence typing (MLST) and clonal complex analysis The sequence type of P. aeruginosa strains was determined following the instructions of Curran et al. (2004). Fragments of seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA and trpE) were amplified by PCR (2.3.3.8) and sequenced (2.3.3.12). The sequence type of the isolate was identified with the sequence definition tool of the P. aeruginosa MLST web site (http://pubmlst.org/paeruginosa/). eBURST analysis was performed to determine the clonal complex to which the isolate belongs using the eBURST software (Feil et al., 2004).
2.3.3.16 Pulsed field gel electrophoresis (PFGE) PFGE was used to separate genomic and plasmid DNA by size with lengths of up to 3 Mbp. The DNA samples were preparated by inoculating 10 ml of TN buffer with colony material from agar plates to a McFarland of 0.3-0.8. After a centrifugation step (4,000 × g, 4 °C, 15 min) the Material and Methods 44
supernatant was discarded and the pellet was resuspended in 500 µl EC buffer. The solution was warmed to 45 °C and RNase A and lysozyme were added to a final concentration of 20 µg/l and 2 mg/ml, respectively. 500 µl pre-warmed InCert agarose were added and mixed with the bacterial suspension by inverting the tube 2-3 times. Immediately after mixing, the suspension was transfered into the slots of a Disposable Plug Mold (Bio-Rad) with 100 µl each. The gel plugs were incubated for 10 min at 4 °C until the plugs were solid. The plugs were incubated in a thermoshaker for 1 h at 37 °C and 300 rpm in 1 ml EC lysis buffer. Afterwards, the lysis buffer was removed and the plugs were incubated 18-24 h at 50 °C and 300 rpm with proteinase K with a final concentration of 1 mg/ml. After the incubation the buffer was removed and the plugs were washed two times in TE buffer with 1 mM AEBSF for 2 h at 37 °C and 300 rpm, followed by two washing steps with TE buffer for 1 h. After washing, the plugs were stored in ES buffer overnight. As the next step the plugs were incubated two times for 15 min in TN buffer, followed by incubation in the specific buffer for the restriction enzyme supplied by the manufacturer. For plasmid PFGE, the DNA was restricted with Nuclease S1, which linearizes plasmid DNA. For chromosomal localization studies, the DNA was restricted with I-Ceu I, which recognizes a highly conserved region in rrn genes (Liu et al., 1993). After restriction the plugs were stored in ES buffer at 4 °C. PFGE was performed by inserting the gel plugs into the slots of a 1 % SeaKem agarose gel and the gel was run with switch times from 1-200 s at 6 V/cm with an angle of 120 ° for 12-36 h in TBE buffer (Bio-Rad). The DNA bands were visualized under UV illumination and the gel was subjected to Southern blotting, if applicable.
TN buffer 1 M NaCl 10 mM Tris/HCl, pH 7.6 pH 7.6
EC buffer 1 M NaCl 6 mM Tris/HCl, pH 7.6 100 mM EDTA 0.5 % (w/v) Brij 58 0.2 % (w/v) Sodium deoxycholate 0.5 % (v/v) Sodium lauroyl sarcosinate pH 7.6
ES buffer 0.5 M EDTA 1 % (v/v) Sodium lauroyl sarcosinate
Material and Methods 45
TE buffer 10 mM Tris/HCl, pH 7.5 1 mM EDTA
2.3.3.17 Southern Blotting and hybridization with gene specific probes For Southern Blotting experiments, DNA fragments were separated by PFGE (2.3.3.16) and the gel was placed onto a positively charged nylon membrane, previously wetted with SSCx2 buffer. The gels were covered with 0.25 M HCl for depurination and a vacuum was applied for 20 min. Afterwards the HCl was removed and the gel was covered with 1 M NaOH. Transfer of DNA to the membrane was achieved by vacuum application for 90 min. The membrane was washed two times in SSCx2 buffer and dried for 30 min at 120 °C. Gene specific digoxigenin-labeled probes were produced by using the PCR DIG Probe Synthesis kit (Roche) with gene-specific oligonucleotides following the manufacturer’s instructions. The membrane was incubated for 30 min at 42 °C in hybridization buffer. 10 µl of the DIG PCR product were added to 40 ml of hybridization buffer and the membrane was incubated with the probe overnight at 42 °C with gentle shaking. After probe incubation the blot was washed two times in washing buffer 1 at room temperature for 5 min, followed by two washing steps in washing buffer 2 at 65 °C for 15 min. Afterwards the membrane was incubated in DIG washing buffer for 2 min and in DIG blocking buffer for 30 min with gentle shaking. After blocking, the blot was incubated with 4 µl Anti-DIG-AP in 40 ml DIG blocking buffer for 30 min. The membrane was washed two times with DIG washing buffer for 15 min and transfered into DIG substrate buffer. The phosphatase reaction was started by plating 4 µl of CSPD in 0.4 ml DIG substrate buffer evenly over the membrane. Chemiluminescence signals were detected with HyperFilmTM autoradiography films (GE Healthcare).
SSC×20 buffer 3 M NaCl 0.3 M Sodium citrate pH 7.0
DIG maleate buffer 0.1 M Maleic acid 0.15 M NaCl
Blocking stock 10 % (w/v) Blocking Reagent (Roche) in DIG Maleate buffer
Material and Methods 46
Hybridization buffer 50 % (v/v) Formamide 2 % (v/v) Blocking stock 0.1 % (v/v) Sodium lauroyl sarcosinate 0.02 % (v/v) SDS in SSC×5 Washing buffer 1 0.1 % (v/v) SDS in SSC×2
Washing buffer 2 0.1 % (v/v) SDS in SSC×0.5
DIG washing buffer 0.3 % (v/v) Tween20 in DIG maleate buffer
DIG blocking buffer 1 % (v/v) Blocking stock in DIG maleate buffer
DIG substrate buffer 0.1 M Tris, pH 9.5 0.1 M NaCl
0.05 M MgCl2
2.3.4 Biochemical methods
2.3.4.1 Purification of β-lactamases by fast protein liquid chromatography (FPLC) E. coli TOP10 cells expressing a -lactamase were grown in 4 l LB medium at 37 °C for 18 h and harvested by centrifugation (3,600β × g, 4 °C, 30 min). Cell pellets were resuspended in 50 ml buffer H or 0.1 -lactamase. Metallo- - lactamase-producingM sodium cells phosphate were resuspended buffer, depending in buffer on Hthe while expressed Ambler β class D -lactamaseβ producing cells were resuspended in sodium phosphate buffer. The cells were disruptedβ by sonication at 4 °C. The lysates were cleared by centrifugation (48.400 × g, 4 °C, 30 min), filtered through a Filtropur S 0.2 µm filter (Sarstedt) and desalted with a HiPrep 26/10 desalting column (GE Healthcare) using the Äkta Pure automated FPLC system (GE Healthcare). The column was previously equilibrated with buffer H or 0.1 M phosphate buffer. After desalting, the protein- containing fractions were loaded onto a 5 ml HiTrap SP HP ion exchange column (GE Healthcare) at a flow rate of 2 ml/min, previously equilibrated with buffer H or 0.1 M phosphate buffer. The column was washed with 10 ml of buffer H and bound proteins were eluted using a linear NaCl gradient (0 to 1 M). Fractions containing high amounts of protein were loaded onto a Material and Methods 47
HiPrep 16/60 S-200 HR gel filtration column (GE Healthcare) equilibrated with buffer H or 0.1 M phosphate buffer, both supplemented with 0.15 M NaCl at a flow rate of 0.8 ml -lactamase containing fractions were identified by incubating 5 µl of eluate with 5 µl of a/min. 1 mM β solution of the chromogenic cephalosporin nitrocefin. A color change from yellow to red indicated the -lactamase in the sample. Fractions that contained high amounts -lactamase presenceactivity were of a pooled, β immediately frozen in liquid nitrogen and stored at -80 °C. of β
Buffer H 50 mM HEPES
50 µM ZnSO4 pH 7.5
Sodium phosphate buffer 0.1 M Sodium phosphate pH 5.9
2.3.4.2 SDS polyacrylamide gel electrophoresis Separation of proteins based on their molecular weight was performed as described by Laemmli (1970). Protein samples were mixed with SDS sample buffer and were incubated at 100 °C for 10 min. 15-20 µl of the sample were loaded onto a 4 % acrylamide stacking gel and seperated by gel electrophoresis in an 11 % acrylamide running gel. Electrophoresis was performed at a voltage of 20-150 V for 1-2 h in SDS running buffer. The gels were stained with Coomassie Brilliant blue after completion of electrophoresis. The PageRuler Plus Prestained standard (Thermo Scientific) served as a size marker.
SDS sample buffer 10 % (w/v) SDS 5 % (v/v) -Mercaptoethanol
0.5 % (w/v) βBromphenol blue 250 mM Tris/HCl, pH 6.8 50 % (v/v) Glycerol
4 % stacking gel 0.5 M Tris/HCl pH 6.8 4 % (v/v) Acrylamide/Bisacrylamide (37.5 : 1) 0.4 % (w/v) SDS 0.06 % (v/v) TEMED 0.1 % (w/v) APS
Material and Methods 48
11 % running gel 0.5 M Tris/HCl, pH 8.8 11 % (v/v) Acrylamide/Bisacrylamide (37.5 : 1) 0.1 % (w/v) SDS 0.04 % (v/v) TEMED 0.1 % (w/v) APS
SDS running buffer 25 mM Tris/HCl, pH 8.3 250 mM Glycine 0.1 % (w/v) SDS
2.3.4.3 Isoelectric focussing -lactamases cell- - lactamasesFor determination were subjected of the to isoelectric isoelectric point focus ofsing. β Cell free extractsfree were extracts prepared or purifiedfrom 10 ml β of an overnight culture. The cells were disrupted by vigorous shaking with glass beads using the PowerLyzer 24. The lysate was cleared by centrifugation (16,000 × g, 5 min). For isoelectric focusing 2 µl of the supernatant or 2 µl of purified enzyme solution were applied onto a CleanGel IEF gel (GE Healthcare) together with reference standards. The gel was cooled to 7 °C during the procedure and was run for 30 min at 500 V, 20 mA and 5 W, for 90 min at 1700 V, 20 mA and 25 W and for 30 min at 2000 V, 20 mA and 30 -lactamases were visualized by adding 2 ml of
1 mM Nitrocefin spread evenly over the gel. W. β
2.3.4.4 Determination of kinetic parameters of purified β-lactamases -lactam antibiotics was monitored by measuring the absorbance changes
Hydrolysis of β -lactam ring. All measurements were performed at 25 °C in bufferresulting H orfrom buffer the openingP in a total of the volume β of 500 µl. - lactam hydrolysis monitoring with an Eppendorf BioSpectrometerThe following wavelenghts (Eppendorf): were used for β
235 nm Penicillin G, Ampicillin, Piperacillin 260 nm Oxacillin, Cefoxitine, Ceftazidime, Cefotaxime 300 nm Imipenem, Meropenem, Ertapenem 320 nm Aztreonam
The substrate concentrations ranged from 0.5 to 1,600 µM, depending on hydrolytic efficiency. Hydrolysis was started by enzyme addition and the reaction was monitored over 10 min with Material and Methods 49
enzyme concentrations ranging from 0.01 to 0.2 µM. For each substrate, the molar extinction -Beer law. coefficient (ε) was determined using the Lambert = ℓ
퐴 휖 ∙ 푐 ∙ A: absorption c: concentration
ℓ: path length The initial rate slopes were calculated from the the reactions linear phase with linear regression using the GraphPad Prism 6 software. The initial velocity of the reaction was determined with a modified Lambert-Beer law and the following formula:
V0 = ℓ 훥퐴
휀 ∙ ∙ ∆푡 V0: initial reaction velocity A change per time
Δ /Δt: absorbtion coefficient of the substrate ε:: molarpath length extinction ℓ The Vmax and Km kinetic parameters were determined with nonlinear regression using the
Michaelis-Menten equitation with the GraphPad Prism 6 software. The turnover number kcat was determined by dividing Vmax by the enzyme concentration.
Buffer H 50 mM HEPES
50 µM ZnSO4 pH 7.5
Buffer P 0.1 M Sodium phosphate
50 mM NaHCO3 pH 7.0
Material and Methods 50
2.3.5 In silico methods
2.3.5.1 In silico DNA and amino acid sequence analysis Computational DNA sequence analysis was performed using various bioinformatic tools. In silico restriction, ligation, cloning and comparison of DNA and protein sequences were performed using the Clone Manager 5 software (Sci-Ed). Bacterial promotor structures were analyzed using the online web tools PromoterHunter (Klucar et al., 2010), BPROM (Solovyev & Salamov, 2011) and SCOPE (Carlson et al., 2007). Integron promoters were analyzed following the classifications of Papagiannitsis et al. (2009). Annotation of DNA sequences was performed using RAST (Aziz et al., 2008; Overbeek et al., 2014) and by manual annotation using BLAST (http://blast.ncbi.nlm.nih.gov) and the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/). Graphical alignment of large DNA sequences was performed using Mauve (Darling et al., 2004; Darling et al., 2010). Prediction of N-terminal signal peptide sequences was done using the SignalP server (Petersen et al., 2011). Circular views of plasmids were constructed using DNAPlotter (Carver et al., 2009). Inc-typing of plasmid sequences was performed using the web-based PlasmidFinder 1.2 software (Carattoli et al., 2014).
2.3.5.2 Phylogenetic analysis of β-lactamases Amino acid sequences were aligned using the ClustalW2 algorithm in MEGA6 software (Tamura et al., 2013). Phylogenetic trees based on alignments were constructed using the neighbour- joining method with 1000 times bootstrapping and the Dayhoff model in MEGA6. Cluster analysis was performed using the Ctree software (Archer & Robertson, 2007).
2.3.5.3 Tertiary structure modelling Protein structure modelling based on amino acid homology was done using the SWISS-MODEL webserver (Arnold et al., 2006; Guex et al., 2009; Kiefer et al., 2009; Biasini et al., 2014). Protein models were visualized using PyMol (http://www.pymol.org/pymol).
2.3.5.4 SDS-PAGE analysis Relative quantification of bands detected in SDS gels was performed using the GelDoc XR+ software (Bio-Rad).
Results 51
3 Results
The ongoing spread and diversification of carbapenemases in Gram-negative pathogens is a major clinical problem. At the German National Reference Laboratory for multidrug-resistant Gram-negative bacteria, three strains attracted attention as the molecular basis of carbapenem resistance was could not be determined in the routine diagnostic process. It was suspected that these isolates harbour novel carbapenemases.
3.1 The search for novel carbapenemases
3.1.1 Identification of IMP-31 in Pseudomonas aeruginosa NRZ-00156 P. aeruginosa NRZ-00156 was found to be highly resistant to carbapenems in routine diagnostics and showed a metallo- -lactamase phenotype. As all PCRs for MBL-type genes were negative, it was suspected that the βisolate harboured a novel MBL. The isolate was analyzed phenotypically by a modified Hodge Test and an EDTA-CDT to ensure the production of a metallo- -lactamase. The results of these tests are shown in Figure 3.1. The modified Hodge Test indicatedβ the production and secretion of a carbapenemase, as the indicator strain was able to grow along the streak of P. aeruginosa NRZ-00156. The EDTA-CDT was clearly positive with an increase in the inhibition zone diameter of 10 mm for imipenem/EDTA and 6 mm for meropenem/EDTA, while the control showed an increase of only
Figure 3.1 Modified Hodge Test and EDTA-CDT of P. aeruginosa NRZ-00156. (A) Modified Hodge Test. The indicator strain E. coli ATCC 25922 was plated on an MH2 agar plate and P. aeruginosa NRZ-00156 was streaked between disks containing imipenem (IPM), meropenem (MEM) and ertapenem (ETP). An invaginating growth of the indicator strain along the test strain streak is highlighted by white arrows. (B) EDTA-CDT. P. aeruginosa NRZ-00156 was plated on an MH2 agar plate. Carbapenem disks were placed in duplicate and EDTA was added to one of the disks. A blank disk with EDTA served as a control. Results 52
4 mm. Consequently, the test indicated the production of an MBL by the isolate. For more detailed resistance analysis, the minimal inhibitory concentrations (MIC) for various -lactams were determined. The results of the MIC analysis are shown in Table 3.1. According toβ EUCAST criteria, the isolate was resistant to piperacillin, piperacillin/tazobactam, cefepime and ceftazidime -lactams not covered by the EUCAST criteria as not commonly used for therapy . againstRegarding P. aeruginosa the β due to intrinsic resistance, the isolate showed MICs that often exceeded the detection range. Susceptibility was detected only for the monobactam aztreonam. With MICs higher than 32 mg/l, the isolate was resistant to the carbapenems imipenem, meropenem and doripenem which was in accordance with the production of a potent carbapenemase. To ensure that the isolate did not harbour a known carbapenemase gene that was accidentally not detected in routine diagnostics, a PCR screening on VIM, IMP, NDM, KHM, SPM, GIM, SIM, DIM, AIM and FIM-type carbapenemase genes was performed. Surprisingly, a
Table 3.1 β-lactam MICs of P. aeruginosa NRZ-00156. Shown are the MICs detected by Etest strips and their interpretation according to EUCAST criteria (R, resistant; I, intermediate, S, susceptible).
P. aeruginosa Interpretation according Antibiotic NRZ-00156 to EUCAST criteria
Ampicillin >256a -b
Ampicillin-sulbactam >256 -
Piperacillin 96 R
Piperacillin-tazobactam 64 R
Amoxicillin >256 -
Amoxicillin-clavulanate 48 -
Temocillin >1024 -
Cephalotin >256 -
Cefuroxime >256 -
Cefoxitin >256 -
Cefotaxime >256 -
Ceftriaxone >256 -
Cefepime >256 R
Ceftazidime >256 R
Imipenem >32 R
Meropenem >32 R
Doripenem >32 R
Ertapenem >32 -
Aztreonam 8 S aThe MIC was higher than detectable by Etest strips, which usually have a concentration range of up to 256 mg/l. bNo clinical MIC EUCAST-breakpoint data are available for these antibiotics for P. aeruginosa. Results 53
PCR with the oligonucleotides IMP-A and IMP-B (Table 2.3) yielded an amplificate for IMP-type genes with a size of approximately 600 bp (data not shown). As the oligonucleotides IMP-A and
IMP-B are degenerate and bind to a large number of blaIMP-type genes, the amplificate was sequenced and showed distinct differences to all other known blaIMP sequences that were publicly available. To sequence the whole open reading frame (ORF) of the potentially new blaIMP variant, a combination of oligonucleotides for conserved class 1 integron regions and blaOXA-10-like genes was used for PCRs, as it is known that blaIMP genes are often associated with blaOXA genes within class 1 integron structures. PCRs with the oligonucleotide combinations 5’CS/IMP-B and
IMP-A/OXA-10B (Table 2.3) yielded amplificates that covered the whole blaIMP ORF and a few hundred base pairs of the flanking genetic environment. The ORF had a size of 738 bp and coded for a 245 amino acid protein. On nucleotide level, the sequence showed a homology of 86 % to blaIMP-8 and blaIMP-24. With regard to blaIMP-1, it showed a homology of only 83 %. On amino acid level, the novel IMP variant showed a homology of 84.1 % to IMP-8 and 83.7 % to IMP-2, IMP-19, IMP-20 and IMP-24. With only 80.0 % homology, IMP-31 was the most divergent IMP-type enzyme relative to the reference enzyme IMP-1. The nucleotide and protein sequences were -lactamase numbering institution (K. Bush & G. Jacoby, Lahey submittedClinic Medical to the Centre, international Burlington, β U.S.; http://www.lahey.org/studies) and the enzyme was assigned as IMP-31. The nucleotide sequence of blaIMP-31 was submitted to the NCBI database (accession number KF148593.1). Shortly after, the amino acid sequence of another novel IMP-type enzyme, IMP-35, was published by another working group and showed a homology of 96.7 % to IMP-31, making IMP-35 the current next nearest relative. An alignment of the amino acid sequences of IMP-31, IMP-35 and IMP-1 is shown in Figure 3.2. Consisting of 245 amino
Figure 3.2 Amino acid sequence alignment of IMP-31, IMP-35 and IMP-1. The highly conserved zinc binding ligands of IMP-type enzymes are marked with asterisks. Results 54
acid residues, IMP-31 lacked one C-terminal amino acid compared to IMP-35 and IMP-1. In contrast to all other known IMP variants, where the C-terminus is mostly formed by a KKXSXPSX motif, the C-terminal amino acid sequence of IMP-31 was KNHHSPK and therewith showed a significant difference. IMP-31 showed 54 amino acid substitutions compared to IMP-38, resulting in an identity of only 78.2 % which is highest diversity of all known IMP variants compared to each other (data not shown). IMP-31 showed no alterations of the highly conserved zinc binding ligands of subclass B1 MBLs. To correlate IMP-31 with all other IMP-type enzymes with publicly available amino acid sequences, a phylogenetic tree was constructed and this tree is shown in Figure 3.3. Cluster analysis using the CTree software clustered the enzymes into thirteen groups, the IMP-1 (including IMP-3, IMP-4, IMP-6, IMP-10, IMP-25, IMP-26, IMP-30, IMP-34, IMP-38, IMP-40 and IMP-42), IMP-2 (including IMP-8, IMP-19, IMP-20 and IMP-24), IMP-5 (including IMP-7, IMP-15, IMP-28 and IMP-43), IMP-9 (including IMP-45), IMP-11 (including IMP-21, IMP-41 and IMP-44), IMP-12 (including only IMP-12), IMP-13 (including IMP-33 and IMP-37), IMP-14 (including
Figure 3.3 Phylogenetic analysis of IMP-31. The tree was constructed based on aligned amino acid sequences of all 42 IMP-type MBLs with publicly available sequences after removal of their N-terminal signal peptides. Construction was performed using the neighbour-joining method with 1000 times bootstrapping and the Dayhoff model. Replicate tree percentages during bootstrapping are shown next to the branches. Clusters were analyzed using the C-tree algorithm and are indicated by parenthezised numbers. Scale: 0.02 substitutions per site. Results 55
IMP-32 and IMP-48), IMP-16 (including IMP-22), IMP-18 (including only IMP-18), IMP-27 (including only IMP-27), IMP-29 (including only IMP-29) and IMP-31 (including IMP-35) groups. The analysis showed that IMP-31 and IMP-35 formed a cluster that showed the highest diversity to any other IMP-type enzyme cluster. To acquire more information on the isolate P. aeruginosa NRZ-00156 and to be able to classify the isolate in an epidemiological context, the MLS type of the isolate was determined by amplification and sequencing of seven P. aeruginosa housekeeping genes. The sequence types and the corresponding MLS type were determined using the sequence definition tool of the P. aeruginosa MLST web site (http://pubmlst.org/paeruginosa/). The results are summarized in Table 3.2. Analysis of the allele types showed that P. aeruginosa NRZ-00156 expressed an allelic profile consistent with ST235, which belongs to the clonal complex CC235.
Table 3.2 MLS typing of P. aeruginosa NRZ-00156. Listed are the seven P. aeruginosa MLST housekeeping genes and the corresponding allele types of P. aeruginosa NRZ-00156.
Gene acsA aroE guaA mutL nuoD ppsA trpE allele type 38 11 3 13 1 2 4
3.1.2 Identification of OXA-233 in Citrobacter freundii NRZ-02127 C. freundii NRZ-02127 attracted attention in routine diagnostics, as the isolate showed susceptibility to oxyimino-cephalosporins but elevated resistance to carbapenems, which was -lactamase genes were inhibited by clavulanic acid. As PCRs for the most common class-lactamase. A and D β negative,To ensure it the was production suspected of that a carbapenemase the isolate harboured by C. freundii a novel NRZ β -02127, the isolate was analyzed by a modified Hodge Test, which is shown in Figure 3.4. The growth of the indicator strain along the streak of C. freundii NRZ-02127 indicated a carbapenemase secretion by the isolate. To
Figure 3.4 Modified Hodge Test of C. freundii NRZ-02127. The indicator strain E. coli ATCC 25922 was plated on an MH2 agar plate and C. freundii NRZ-02127 was streaked between disks containing imipenem (IPM), meropenem (MEM) and ertapenem (ETP). An invaginating growth of the indicator strain along the test strain streak is highlighted by white arrows. Results 56
further characterize the resistance phenotype of the iso -lactams were determined. The results are summarized in Table 3.3. Thelate, isolate the MICs was forresistant various to β penicillins and showed a noticable inhibition by clavulanic acid, but not by sulbactam or tazobactam. It was resistant to cefuroxime, but showed very low MICs for oxyimino-cephalosporins and was interpreted as susceptible to cefepime and intermediate to ceftazidime according to the EUCAST criteria. Carbapenem MICs were interpreted as intermediate for imipenem and meropenem and resistant for doripenem and ertapenem. To identify the molecular basis of this resistance phenotype, shotgun cloning experiments were performed. The experiment -lactam resistant clone that showed the same resistance profile as C. freundii NRZ-02127s yielded with a increased β resistance to carbapenems but not to oxyimino- cephalosporins. Sequencing of the insert of the recombinant plasmid pMB3002 revealed an 801-bp ORF coding for a protein consisting of
Table 3.3 β-lactam MICs of C. freundii NRZ-02127. Shown are the MICs detected by Etest strips and their interpretation according to EUCAST criteria (R, resistant; I, intermediate, S, susceptible).
Interpretation according Antibiotic C. freundii NRZ-02127 to EUCAST criteria
Ampicillin >256a R
Ampicillin-sulbactam >256 R
Piperacillin >256 R
Piperacillin-tazobactam >256 R
Amoxicillin >256 R
Amoxicillin-clavulanate 64 R
Temocillin 64 -b
Cephalotin >256 -
Cefuroxime 24 R
Cefoxitin >256 -
Cefotaxime 0.75 S
Ceftriaxone 0.75 -
Cefepime 0.38 S
Ceftazidime 1.5 I
Imipenem 3 I
Meropenem 6 I
Doripenem 3 R
Ertapenem >32 R
Aztreonam 0.5 S
aThe MIC was higher than detectable by Etest strips, which usually have a concentration range of up to 256 mg/l. bNo clinical MIC EUCAST-breakpoint data are available for these antibiotics for Enterobacteriaceae according. Results 57
266 amino acids. A BLAST homology search revealed that the sequence was 99.8 % identical to the blaOXA-17 gene and 99.75 % to the blaOXA-19 gene. Sequence analysis showed that the gene exhibited a single nucleotide substitution compared to the blaOXA-17 gene at position 349 from guanine to tymine. Compared to the blaOXA-10 gene, the sequence showed an additional substitution at position 218 from alanine to guanine. The sequence of the novel blaOXA gene was -lactamase numbering institution and the encoded enzyme was submittedassigned as to OXA the- 233.international The nucleotide β sequence of blaOXA-233 was submitted to the NCBI database (accession number KJ657570.1). OXA-233 was compared with the two next nearest relatives OXA-17 and OXA-10 and an alignment of the amino acid sequences is shown in Figure 3.5. The amino acid sequences of OXA-233 and OXA-17 differed in a valine to phenylalanine substitution at the highly conserved position 117. Compared to OXA-10, OXA-233 exhibited an additional point mutation at position 73 from asparagine to serine, while this mutation is also found in OXA-17. This resulted in identities of 99.2 % to OXA-10 and 99.6 % to OXA-17. The highly conserved STFK-motif at positions 67 to 70 which includes the active site serine was not altered in OXA-233. No MLS typing scheme existed for C. freundii at that time, and the MLS type of the isolate could not be determined. As class D -lactamases are a very heterogenous group of enzymes and as OXA-233 was closely relatedβ solely to enzymes of the OXA-10 subgroup, no further phylogenetic analysis was performed.
Figure 3.5 Amino acid sequence alignment of OXA-233, OXA-17 and OXA-10. Highly conserved regions of class D -lactamases are highlighted. -lactamase is marked with an asterisk.
β The active site serine residue of class D β
Results 58
3.1.3 Identification of KHM-2 in Pseudomonas aeruginosa NRZ-03096 Like P. aeruginosa NRZ-00156, P. aeruginosa NRZ-03096 attracted attention in routine diagnostics as the isolate exhibited high carbapenem resistance that was inhibited by EDTA, indicating an MBL production. As all diagnostic PCRs were negative, the isolate was suspected to harbour a novel MBL. The isolate was analyzed phenotypically for metallo- -lactamase production by a modified
Hodge Test and an EDTA-CDT. The results are shown inβ Figure 3.6 and the isolate indicated a carbapenemase secretion in the Hodge Test. MBL production was indicated by increased inhibition zone diameters of 13 mm for imipenem/EDTA (10 to 23 mm) and 19 mm (6 to 25 mm) for meropenem/EDTA, while the control showed an inhibition zone diameter of 19 mm. -lactams and interpretation according the EUCAST criteria
Determinationshowed that the of isolate the MICswas susceptible for β to piperacillin and piperacillin/tazobactam and resistant to cefepime and ceftazidime with MICs of 64 and >256 mg/l, respectively (Table 3.4). The isolate was susceptible to imipenem with an MIC of only 3 mg/l, while it was intermediate for meropenem and resistant to doripenem with MICs of 6 and 8 mg/l, respectively. The MIC for ertapenem was >32 mg/l. Regarding the antibiotics with no EUCAST breakpoints, the isolate showed high MICs for ampicillin, amoxicillin and most cephalosporins with an MIC of >256 mg/l. Although the carbapenem MICs were not as high as for the IMP-31 containing isolate P. aeruginosa NRZ-00156, the observed values indicated the presence of a carbapenem resistance mechanism. To exclude that the isolate harboured a known carbapenemase gene that was not detected in
Figure 3.6 Modified Hodge Test and EDTA-CDT of P. aeruginosa NRZ-03096. (A) Modified Hodge Test. The indicator strain E. coli ATCC 25922 was plated on an MH2 agar plate and P. aeruginosa NRZ-03096 was streaked between disks containing imipenem (IPM), meropenem (MEM) and ertapenem (ETP). A growth of the indicator strain along the test strain streak is highlighted by white arrows. (B) EDTA-CDT. P. aeruginosa NRZ-03096 was plated on an MH2 agar plate. Carbapenem disks were placed in duplicate and EDTA was added to one of the disks. A blank disk with EDTA served as a control. Results 59
Table 3.4 β-lactam MICs of P. aeruginosa NRZ-03096. Shown are the MICs detected by Etest strips and their interpretation according to EUCAST criteria (R, resistant; I, intermediate, S, susceptible).
P. aeruginosa Interpretation according Antibiotic NRZ-03096 to EUCAST criteria
Ampicillin >256a -b
Ampicillin-sulbactam >256 -
Piperacillin 4 S
Piperacillin-tazobactam 2 S
Amoxicillin >256 -
Amoxicillin-clavulanate >256 -
Temocillin >1024 -
Cephalotin >256 -
Cefuroxime >256 -
Cefoxitin >256 -
Cefotaxime >256 -
Ceftriaxone >256 -
Cefepime 64 R
Ceftazidime >256 R
Imipenem 3 S
Meropenem 6 I
Doripenem 8 R
Ertapenem >32 -
Aztreonam 1.5 I
aThe MIC was higher than detectable by Etest strips, which usually have a concentration range of up to 256 mg/l. bNo clinical MIC EUCAST-breakpoint data are available for these antibiotics for Enterobacteriaceae. routine diagnostics, a PCR screening on VIM, IMP, NDM, KHM, SPM, GIM, SIM, DIM, AIM and FIM- typ MBL genes was performed, but all PCRs were negative. To identify the putative novel carbapenemase gene, a shotgun cloning approach was taken. As experiments using genomic DNA that was digested with HindIII, EcoRI, XhoI and BamHI did not yield any recombinant clones, MboI was used for restriction. The partially digested genomic DNA was then ligated with the BamHI-digested pBK-CMV vector, as the MboI and BamHI overhangs (GATC) are compatible. Finally, the MboI experiments yielded a clone with increased resistance for carbapenems and the 4575-bp insert of the contained recombinant plasmid pMB3013 was sequenced using oligonucleotides listed in Table 2.3. It harboured a 726-bp ORF that coded for a 241 amino acid protein. On both nucleotide and protein level the sequences showed a homology of only 74.3 %
Results 60
Figure 3.7 Amino acid sequence alignment of KHM-2 and KHM-1. The residues known as subclass B1 zinc binding residues of are marked with asterisks.
to blaKHM-1, coding for the metallo- -lactamase KHM-1. The sequence of the novel blaKHM gene was β -lactamase numbering institution and the encoded enzyme was submittedassigned as to KHMthe international-2. An alignment β of the amino acid sequences of KHM-2 and KHM-1 is shown in Figure 3.7. Compared to KHM-1, KHM-2 showed no alterations in the highly conserved zinc binding residues, but exhibited a threonine to aspartic acid substitution at position 100, which is part of the conserved HXHXD zinc binding motif. With only 74.3 % homology, KHM-2 showed one of the greatest distances to the next nearest relative within the Ambler subclass B1. As KHM-2 and KHM-1 were the only members of the KHM group, no further phylogenetic analysis was performed. Compared zo other subclass B1 enzymes, KHM-2 showed similarities of only 54 % to IMP-1, 29 % to VIM-2 and 29 % to NDM-1. To acquire more information on the isolate P. aeruginosa NRZ-03096 and to be able to classify the isolate in an epidemiological context, the MLS type of the isolate was determined by amplification and sequencing of seven P. aeruginosa housekeeping genes. Like for P. aeruginosa NRZ-00156, the sequence types and the corresponding MLS type were determined using the sequence definition tool of the P. aeruginosa MLST web site (http://pubmlst.org/paeruginosa/). The allele types are summarized in Table 3.5 and showed that P. aeruginosa NRZ-03096 expressed an unknown allelic profile. This was based on a point mutation and two insertions in the 3´ region of the aroE gene. The closest match for the aroE sequence type was type 5, resulting in MLST 395 beeing the nearest relative to the sequence type expressed by P. aeruginosa NRZ-03096.
Table 3.5 MLS typing of P. aeruginosa NRZ-03096. Listed are the seven P. aeruginosa MLST housekeeping genes and the corresponding allele types (ST) of P. aeruginosa NRZ-03096. Parts of the results were obtained by Hoffmann (2013).
Gene acsA aroE guaA mutL nuoD ppsA trpE allele type 6 closest match: 5 1 1 1 12 1 Results 61
3.2 Analysis of the genetic environment of blaIMP-31, blaOXA-233 and blaKHM-2 -lactamase genes can have a significant influence on the gene expressionThe genetic level environment and the ability of β of the gene to be horizontally transferred to other bacteria. To identify genetic structures like integrons or transposable elements, the genetic context of the three novel carbapenemase genes blaIMP-31, blaOXA-233 and blaKHM-2 was analyzed by cloning and sequencing techniques.
3.2.1 Genetic environment of blaIMP-31
To further explore the genetic environment of the blaIMP-31 gene, a shotgun cloning approach was performed. Shotgun cloning experiments with MboI finally yielded one single E. coli TOP10 clone that showed increased resistance for carbapenems. The insert of the contained pBK-CMV derivative plasmid pMB3011 was sequenced and the 2767-bp insert covered the whole blaIMP-31
ORF. In addition, it covered the neighboring blaOXA-10-like gene which was identified as blaOXA-35. However, the shotgun cloning approach failed to provide significant additional information on the surrounding regions, as apart from the two genes mentioned, the insert only covered 330 bp of the 3´region of an intI1 gene upstream of the blaIMP-31 gene and 193 bp of a sequence with high similarities to an aminoglycoside-acetyltransferase encoding gene downstream of the blaOXA-35 gene. To further analyze the genetic environment of blaIMP-31, a genome walking approach was chosen. Using the Universal GenomeWalker 2.0 kit (Clontech), a DNA fragment with a size of approximately 6 kb was amplified and sequenced using oligonucleotides listed in Table 2.3. By combination of the sequences obtained from PCRs and genome walking it was possible to assemble 4.8 kb of the genomic environment of the blaIMP-31 gene. A schematic of the genetic environment of blaIMP-31 is shown in Figure 3.8. Sequence analysis showed that the gene was part of a disrupted class 1 integron as the first gene cassette directly after the attI site. Further downstream, gene cassettes containing blaOXA-35, aac(6’)-Ib, aac(3)-Ic and aphA15 genes were identified. Downstream of the aphA15 gene cassette, the integron was disrupted by a transposon-like structure, consisting of a tniC gene, which encodes for a site-specific
Figure 3.8 Genetic environment of blaIMP-31 in P. aeruginosa NRZ-00156. Conserved integron structures are shown in grey. The sequences of the integron promoters PcH2 and the inactive P2 are shown below and framed grey. The -35 and -10 boxes are marked with bold letters. Results 62
recombinase and 525 bp of the tnpA gene, coding for the transposase A protein. Consequently, the obtained sequences did not cover the full putative transposon and the missing 3´CS region of the class 1 integron. The sequence was analyzed for direct and inverted repeats that could serve as integration sites for the putative transposon, but as the transposon was not fully covered by the obtained sequence, no repeat regions could be identified that could be associated to the putative transposon. However, a potential repeat region with a high GC content and sequences of multiple identical bases was identified downstream of the aphA15 gene. Analysis of the integron promoter region revealed that the integron gene cassettes were expressed under the control of the hybrid PcH2 promoter, consisting of the perfect -35 box TTGACA and the -10 box TAAGCT, separated by a 17-bp spacer. The P2 promoter exhibited a 14-bp spacer region between the -35 box TTGTTA and the -10 box TACAGT and was missing the insertion of three guanine bases which optimize the spacing in active P2 variants, resulting in a probably weak or inactive P2 promoter.
3.2.2 Genetic environment of blaOXA-233 The pBK-CMV derivative pMB3002 was obtained from shotgun cloning experiments and harboured an insert with a size of 9102 bp. The insert was fully sequenced using oligonucleotides listed in Table 2.3 and sequence analysis revealed that the blaOXA-233 gene was part of a class 1 integron as the second gene cassette. Upstream, an aac(6´)-Ib gene was identified, coding for an aminoglycoside-acetyltransferase. Downstream, the blaOXA-233 gene was followed by the conserved 3´CS region of the integron, consisting of the genes qacEΔ1 and sul1. A schematic of the genetic environment of blaOXA-233 is shown in Figure 3.9. In silico promoter analysis revealed that the integron cassettes were under the control of a strong Pc promoter, combined with an inactive P2 promoter. The Pc promoter exhibited the perfect -35 box TTGACA and the -10 box TAAACT, resulting in a strong promoter. Like in the blaIMP-31 carrying integron, the P2 promoter was inactive with a TTGTTA -35 box and a TACAGT -10 box that were separated by only 14 spacing base pairs. In the sequence covered by the insert of pMB3002, no
Figure 3.9 Genetic environment of blaOXA-233 in C. freundii NRZ-02127. Conserved integron structures are shown in grey. The sequences of the integron promoters PcH2 and the inactive P2 are shown below and highlighted grey. The -35 and -10 boxes are marked with bold letters. Parts of this figure are based on results obtained by Meining (2012). Results 63
transposon or IS structures were identified. Furthermore, no direct or inverted repeats flanking the integron could be found.
3.2.3 Genetic environment of blaKHM-2
To acquire further information on the genetic environment of blaKHM-2, the 4575-bp insert of the recombinant plasmid pMB3013 was fully sequenced using oligonucleotides listed in Table 2.3. A schematic of the genetic environment is shown in Figure 3.10. Upstream of the blaKHM-2 gene, a 930-bp part of an ORF was identified. It showed 74 % identity to a gene coding for a putative transposase of the ISXo2 family. Downstream of the blaKHM-2 gene, an ORF coding for a 262 amino acid protein was identified. A BLAST homology search yielded a single hit that had an identity of 68 % to the putative gene. This sequence was annotated as an aac(3´) gene in the nucleotide database of the National Centre for Biotechnology Information (NCBI) and was found in a Gloeobacter violaceus whole genome sequence and consequently annotated to code for an aminoglycoside-acetyltransferase. Downstream of the putative aac gene, the gene for a putative insE family transposase was identified. As the region contained two putative transposase genes, the sequence was analyzed for direct and inverted repeats. Upstream of the blaKHM-2 gene, a palindromic sequence was identified (CCAATCATATTAATTGGATTGG) that could serve as an insertion site for either the Isxo2 or InsE transposase, but no equivalent repeat was found in the rest of the sequence covered by the pMB3013 insert. The rest of the sequence did not contain any noticable repeat or inverted repeat regions.
In silico promoter analysis of the genetic environment revealed that the promoter of the blaKHM-2 gene was located 52 bp upstream of the ATG triplet and exhibited the -35 box TCGACA and the - 10 box AAATTA with a 17-bp spacing sequence. The sequence covered by the insert of pMB3013 did not contain any integron-like structures associated with the blaKHM-2 gene.
Figure 3.10 Genetic environment of blaKHM-2 in P. aeruginosa NRZ-03096. Putative transposon structure genes are shown as grey arrows. The sequences of the promoter structures upstream of the blaKHM-2 gene are shown below. The promoter of the blaKHM-2 gene is framed grey and the -35 and -10 boxes are marked with bold letters. The ATG triplet of the blaKHM-2 gene is marked with black framed bold letters. Parts of this figure are based on results obtained by Hoffmann (2013). Results 64
As the shotgun cloning approach for KHM-2 was performed using MboI as restriction enzyme for genomic DNA digestion and as digestion of DNA by MboI results in relatively small DNA fragments, it was possible that the sequence of the insert was assembled by ligation of multiple gDNA fragments from different regions of the DNA and did not represent the actual organization in P. aeruginosa NRZ-03096. To verify that the arrangement shown in Figure 3.10 reflects the actual arrangement in the isolate, PCRs that covered the 3`and 5`ends of neighboring ORFs
(isxO2/blaKHM-2; blaKHM-2/aac(3´)-like; aac(3´)-like/insE) were designed and performed with total DNA from P. aeruginosa NRZ-03096 using oligonucleotides listed in Table 2.3. All used combinations of oligonucleotides yielded PCR products of the expected size (data not shown) and this was taken as verification, that the sequence arrangement reflected the actual state in the isolate.
3.3 Localization of blaIMP-31, blaOXA-233 and blaKHM-2 Resistance genes can be chromosome- or plasmid-encoded. Plasmid-encoded genes can be mobilized by conjugation of the plasmid, while chromosome-encoded genes can be mobilized by transconjugable transposons, which is a less effective mechanism of gene distribution than conjugative plasmids. In this context, it was analyzed if the novel carbapenemase genes identified in this thesis were plasmid-encoded or if they were part of the chromosome of the respective isolate.
3.3.1 Localization of blaIMP-31 To identify the localization of the IMP-31 encoding gene, total DNA from P. aeruginosa NRZ-00156 was digested with nuclease S1 and I-CeuI and separated by PFGE. Nuclease S1 cuts circular DNA molecules exactly once, leading to linearization of plasmids and chromosomes. I-CeuI recognizes and digests a 26-bp sequence in bacterial rrn genes which code for the 23S ribosomal subunit. As P. aeruginosa usually harbours four copies of the 23S rDNA that are located exclusively on the chromosome, a digestion with I-CeuI yields four genomic DNA fragments. As the 16S rDNA is also chromosome-located and neighbored to the 23S rDNA, each of the fragments should contain a single copy of an intact 16S rDNA. PFGE analysis after nuclease S1-digestion showed no detectable linearized plasmid bands and indicated that the isolate did not harbour any plasmid that could be the carrier for blaIMP-31 (data not shown). Consequently, it was suggested that the gene was chromosome-located. A digestion with I-CeuI, followed by PFGE yielded four fragments with sizes of approximately 900 kb, 1,000 kb, 1,300 kb and 2,200 kb. The results are shown in Figure 3.11. Southern blotting and hybridization with digoxigenin (DIG)-labeled DNA probes specific for blaIMP-31 and the 16S rDNA
Results 65
Figure 3.11 Localization of blaIMP-31. Total DNA of P. aeruginosa NRZ-00156 was digested with I-CeuI, separated by PFGE and subjected to Southern Blotting. Hybridization was performed with blaIMP-31 and 16S rDNA gene-specific probes. The figure shows the ethidiumbromide-stained PFGE-lanes of the size Marker (M), the total DNA (L) and the corresponding hybridized blots (IMP and 16S). Signals were detected using an Anti-DIG-AP-coupled antibody, the alkaline phosphatase substrate CSPD and autoradiography films. The blaIMP-31 signal and the corresponding 16S signal are indicated with an arrow. was performed and with the 16S probe, four signals at the exact same size as in the EtBr-stained gel were detected, corresponding to the four chromosome fragments. Hybridization with a blaIMP-31 specific probe yielded a weak, but detectable signal at the size of the 2,200 kb fragment.
As the signals detected for blaIMP-31 and the 16S rDNA matched the 2,200 kb fragment detected in
PFGE, it was indicated that the blaIMP-31 gene was chromosome-located in P. aeruginosa NRZ-00156.
3.3.2 Localization of blaOXA-233 As it is known that OXA-type carbapenemases are often plasmid-encoded, transconjugation experiments were performed with the OXA-233 carrying isolate C. freundii NRZ-02127. Finally, -lactam resistant E. coli C600 clone which exhibited the same theresistance experiments profile yielded as the a clinical β isolate with increased resistance to carbapenems but susceptibility to oxyimino-cephalosporins (Table 3.8) and was PCR-positive for blaOXA-233 (data not shown). Both the clinical isolate and the transconjugant were subsequently analyzed by nuclease S1 digestion and PFGE, followed by Southern blotting and hybridization with a blaOXA-233 specific probe. The results of these experiments are shown in Figure 3.12. In PFGE analysis, the isolate C. freundii NRZ-02127 showed three plasmid bands that had a size of
Results 66
Figure 3.12 Localization of blaOXA-233. Total DNA of C. freundii NRZ-02127 and the E. coli C600 OXA-233 transconjugant was digested with nuclease S1, separated by PFGE and subjected to Southern Blotting and hybridization with a blaOXA-233 gene-specific probe. The figure shows the ethidiumbromide-stained PFGE-lanes of the size Marker (M), the total DNA of C. freundii NRZ-02127 (A) and the transconjugant (B). The corresponding hybridized blot is shown to the right. Signals were detected using an Anti-DIG-AP-coupled antibody, the alkaline phosphatase substrate CSPD and autoradiography films. The OXA-233 carrying plasmid band and the corresponding blaOXA-233 signals are indicated with an arrow. approximately 50, 90 and 200 kb. The OXA-233 transconjugant showed only the 50-kb band, indicating that the blaOXA-233 was most likely encoded by this plasmid. Southern blotting, followed by hybridization with a DIG-labeled blaOXA-233 probe showed signals at the size of the
50-kb band. This indicated that the gene was located on this plasmid, as signals for blaOXA-233 were detected at the exactly same height as the 50 kb-band in the PFGE gel lanes.
3.3.3 Localization of blaKHM-2 To identify the localization of the KHM-2-encoding gene, total DNA from P. aeruginosa NRZ-03096 was digested with nuclease S1 and I-CeuI and separated by PFGE as it was performed for P. aeruginosa NRZ-00156. PFGE analysis after nuclease S1-digestion showed no detectable linearized plasmid bands and indicated that the isolate did not harbour any plasmid that could be the carrier for blaKHM-2 (data not shown). A digestion with I-CeuI, followed by PFGE yielded four fragments with sizes of approximately 610 kb, 825 kb, 1,000 kb and 2,200 kb. The results are shown in Figure 3.13. Southern blotting and hybridization with digoxigenin (DIG)- labeled DNA probes specific for blaKHM-2 and the 16S rDNA showed four detectable signals for the 16S-probe that exactly corresponded to the four chromosome fragments detected in PFGE.
Results 67
Figure 3.13 Localization of blaKHM-2. Total DNA of P. aeruginosa NRZ-03096 was digested with I-CeuI, separated by PFGE and subjected to Southern Blotting. Hybridization was performed with blaKHM-2 and 16S rDNA gene-specific probes. The figure shows the ethidiumbromide-stained PFGE-lanes of the size Marker (M), the total DNA (L) and the corresponding hybridized blots (KHM and 16S). Signals were detected using an Anti-DIG-AP-coupled antibody, the alkaline phosphatase substrate CSPD and autoradiography films. The blaKHM-2 signal and the corresponding 16S signal are indicated with an arrow.
Hybridization with a blaKHM-2 specific probe yielded detectable signal at the size of the 2,200 kb fragment. As the signals detected for blaIMP-31 and the 16S rDNA matched the 2,200 kb fragment detected in PFGE, it was indicated that the blaKHM-2 gene was chromosome-located in P. aeruginosa NRZ-03096.
3.4 Impact of IMP-31, OXA-233 and KHM-2 on β-lactam resistance To analyze the effect of expression of IMP-31, OXA-233 and KHM-2 on the resistance against -lactam antibiotics, the encoding genes were cloned into the pBK-CMV vector and the resulting plasmidsβ were transformed into E. coli TOP10. E. coli TOP10 is a K12 determinant that is lacking a functional LacI protein due to a point mutation in the lacI gene. This results in a constitutive expression of the lac operon and other genes that are under the control of a lac promoter. As the expression of genes which are cloned into the MCS of the pBK-CMV vector is controlled by such a promoter, these genes are constitutively expressed in E. coli TOP10. By determination of the minimal inhibitory concentration (MIC) for variou -lactam antibiotics for E. coli TOP10 expressing the genes identified in this study, the influences β of the production of IMP-31, OXA-233 and KHM- -lactam resistance was analyzed in relation to E. coli TOP10 carrying the empty pBK-CMV 2vector on β and not -lactamase. Contrary to the MIC data for the clinical isolates, the data were not producinginterpreted a according β to the EUCAST criteria as these criteria are not applicable to laboratory E. coli K12 determinant strains. Results 68
3.4.1 Impact of IMP-31 on β-lactam resistance To study the impact of production of IMP- -lactam resistance, the encoding gene was cloned into the pBK-CMV vector, yielding the31 recombinant on β plasmid pMB3007. The plasmid was then transformed into E. coli TOP10. The gene coding for the IMP reference enzyme IMP-1 was also cloned into the pBK-CMV vector (yielding pMB3010) and transformed into E. coli TOP10 to serve as a reference. E. coli TOP10 transformed with the pBK-CMV vector was used as a control. The MICs obtained from these experiments are summarized in Table 3.6. Compared to the control strain, IMP-31 producing E. coli TOP10 showed increased resistan - lactams. The MIC for ampicillin was increased over 10-fold from 1.5 mg/l to 16ce mg/l to all and tested 12-fold β for ampicillin/sulbactam (1.0 to 12 mg/l). Piperacillin and piperacillin/tazobactam MICs were only slightly increased from 1.0 mg/l to 3 mg/l and 0.75 mg/l to 3 mg/l, which corresponds to a 3-fold and 4-fold increase. Production of IMP-31 further resulted in a 128-fold increase in the MIC for amoxicillin. The MIC for amoxicillin/clavulanate however was only increased 11-fold.
Table 3.6 β-lactam MICs of E. coli TOP10 transformed with the pBK-CMV vector and IMP-31/IMP-1 expressing E. coli TOP10. MIC increases relative to the control strain (E. coli TOP10/pBK-CMV) are shown in parentheses.
MIC (mg/l) E. coli TOP10/pMB3007 E. coli TOP10/pMB3010 Antibiotic E. coli TOP10/pBK-CMV IMP-31 IMP-1
Ampicillin 1.5 16 (11×) 48 (32×)
Ampicillin-sulbactam 1.0 12 (12×) 48 (48×)
Piperacillin 1.0 3 (3×) 3 (3×)
Piperacillin-tazobactam 0.75 3 (4×) 3 (4×)
Amoxicillin 2 256 (128×) >256 (>128×)
Amoxicillin-clavulanate 1.5 16 (11×) 32 (21×)
Temocillin 8 16 (2×) 32 (4×)
Cephalotin 3 64 (21×) >256 (>85×)
Cefuroxime 2 64 (32×) >256 (>128×)
Cefoxitin 4 >256 (>64×) >256 (>64×)
Cefotaxime 0.032 3 (94×) 16 (500×)
Ceftriaxone 0.047 3 (64×) 32 (681×)
Cefepime 0.023 0.75 (32×) 3 (130×)
Ceftazidime 0.25 32 (128×) >256 (>1024×)
Imipenem 0.19 0.38 (2×) 1.5 (8×)
Meropenem 0.016 0.19 (12×) 0.75 (47×)
Doripenem 0.016 0.19 (12×) 0.5 (31×)
Ertapenem 0.006 0.19 (32×) 1.0 (167×)
Aztreonam 0.094 0.094 (1×) 0.094 (1×) Results 69
Temocillin MICs were only 2-fold increased from 8 mg/l to 16 mg/l. The MIC increases for first and second generation cephalosporins ranged from over 64-fold for cefoxitin (4 to >256 mg/l) to 21-fold for cephalotin (3 to 64 mg/l). Third generation cephalosporin MICs were increased 94-fold for cefotaxime, 64-fold for ceftriaxone, 32-fold for cefepime and 128-fold for ceftazidime. The MIC for imipenem was increased from 0.19 mg/l to 0.38 mg/l, which was only a 2-fold increase, while the meropenem and doripenem MICs were both increased 12-fold from 0.016 mg/l to 0.19 mg/l. With a 32-fold increase, ertapenem showed the highest carbapenem MIC elevation with values of 0.006 and 0.19 mg/l for the control strain and the IMP-31 producing strain, respectively. The expression of IMP-31 had no effect on the MIC for aztreonam. Compared to the IMP-1 producing strain, the MICs of the IMP-31 producing E. coli TOP10 were generally lower with only the MICs for piperacillin and piperacillin/tazobactam beeing 3 mg/l for both strains. Production of IMP-1 led to MICs of 48 mg/l for ampicillin and ampicillin/sulbactam, while the MIC for amoxicillin was similar to the IMP-31 strain. The greatest differences between IMP-1 and IMP-31 were seen for the oxyimino-cephalosporins cefotaxime, ceftriaxone and ceftazidime with MICs of 16 mg/l, 32 mg/l and >256 mg/l, corresponding to a 500-fold, 681-fold and >1024-fold increase relative to the control strain. Carbapenem MICs were elevated 8-fold for imipenem, 47-fold for meropenem, 31-fold for doripenem and 167-fold for ertapenem, showing significantly higher MICs than IMP-31 producing E. coli TOP10. Like for IMP-31, the expression of IMP-1 had no effect on the MIC for aztreonam. In general, the production of IMP- - lactams, although production of the reference enzyme31 led toIMP increased-1 resulted MICs in even for almost higher allMICs. tested β
3.4.2 Impact of OXA-233 on β-lactam resistance
To analyze the impact of OXA- -lactam resistance, the blaOXA-233 was cloned into the pBK-CMV vector, yielding233 theproduction recombinant on β plasmid pMB3006. To serve as a reference, the blaOXA-10 gene was cloned the same way (yielding pMB3026). Both strains were analyzed in MIC studies and the results are shown in Table 3.7. E. coli TOP10 cells transformed with the pBK- -lactam MICs were also determined for the E. coli
C600CMV OXA - vector233 transconjugant served as a control.that carried The the β plasmid pMB3018 from the clinical isolate. E. coli C600 without any plasmid served as a control and the results are shown in Table 3.8. MIC determination showed that the OXA-233 producing strain exhibited elevated -lactam resistance against most tested antibiotics. The pMB3006 harbouring strain showed anβ MIC of >256 mg/l for ampicillin and amoxicillin, resulting in a more than 170-fold increase compared to the control strain. For piperacillin and amoxicillin, MICs of 16 mg/l and >256 mg/l were det -lactam-inhibitor combinations led to significantly decreased MICs, indicating an inhibitionected. βof OXA-233 by sulbactam, tazobactam and clavulanic acid. Regarding cephalosporins,
Results 70
Table 3.7 β-lactam MICs of E. coli TOP10 transformed with the pBK-CMV vector and OXA-233/OXA-10 expressing E. coli TOP10. MIC increases relative to the control strain (E. coli TOP10/pBK-CMV) are shown in parentheses.
MIC (mg/l) E. coli TOP10/pMB3006 E. coli TOP10/pMB3026 Antibiotic E. coli TOP10/pBK-CMV OXA-233 OXA-10
Ampicillin 1.5 >256 >(170×) >256 (>170×)
Ampicillin-sulbactam 1.0 12 (12×) >256 (>256×)
Piperacillin 1.0 16 (16×) >256 (>256×)
Piperacillin-tazobactam 0.75 2 (3×) 24 (32×)
Amoxicillin 2 >256 (>128×) >256 (>128×)
Amoxicillin-clavulanate 1.5 6 (4×) 32 (21×)
Temocillin 8 8 (1×) 12 (1.5×)
Cephalotin 3 3 (1×) 12 (4×)
Cefuroxime 2 2 (1×) 6 (3×)
Cefoxitin 4 4 (1×) 4 (1×)
Cefotaxime 0.032 0.047 (1.5×) 0.125 (4×)
Ceftriaxone 0.047 0.047 (1×) 0.38 (8×)
Cefepime 0.023 0.023 (1×) 0.094 (4×)
Ceftazidime 0.25 0.25 (1×) 0.25 (1×)
Imipenem 0.19 0.25 (1.3×) 0.25 (1.3×)
Meropenem 0.016 0.032 (2×) 0.032 (2×)
Doripenem 0.016 0.064 (4×) 0.094 (6×)
Ertapenem 0.006 0.094 (16×) 0.064 (11×)
Aztreonam 0.094 0.094 (1×) 0.75 (8×) the OXA-233 strain exhibited values of 3 mg/l, 2 mg/l and 4 mg/l, while showing no increase for cephalotin and cefoxitin resistance compared to the control that showed MICs of 3 mg/l and 4 mg/l, respectively. Furthermore, the strain showed only slightly increased resistance to oxyimino-cephalosporins, confirming the resistance phenotype of C. freundii NRZ-02127 (Table 3.3). Carbapenem MICs of the pMB3006 strain were elevated compared to the control strain with a 4-fold increase for doripenem and a 15.7-fold increase for ertapenem. Imipenem and meropenem MICs were elevated only 1.3-fold and 2-fold, respectively. The expression of OXA-233 in E. coli TOP10 had no effect on the resistance to aztreonam. In comparison with the OXA-233 producing strain, the OXA-10 expressing strain exhibited a significantly higher MIC for piperacillin with a value of >256 mg/l, while the ampicillin and amoxicillin MICs were identical for both strains. The strains however differed in the MICs for penicillin-inhibitor combinations as the OXA-10 strain was not inhibted by sulbactam and less inhibited by tazobactam and claculanic acid with MICs of 24 mg/l and 32 mg/l, respectively. The MICs for cephalosporins Results 71
were higher compared to the OXA-233 strain, although the increases were relatively low with values of 0.125 mg/l for cefotaxime or 0.094 mg/l for cefepime. Surprisingly, the OXA-10 strain showed nearly the same MICs for carbapenems as the OXA-233, which showed a 1.3-fold increase for imipenem, a 2-fold increase for meropenem, a 4-fold increase for doripenem and a 16-fold increase for ertapenem. In contrast to OXA-233, the production of OXA-10 led to an MIC increase for aztreonam from 0.094 mg/l to 0.75 mg/l -lactam MICs were also determined for the OXA-233 transconjugant. It showed the same resistance. β profile as the clinical isolate and the pMB3006 strain with high level resistance to penicillins that was inhibited by sulbactam, tazobactam and clavulanic acid and susceptibility to oxyimino-cephalosporins. Like in the other OXA-233 strain, carbapenem MICs were distinctly elevated except for imipenem, where the MIC was not increased compared to the E. coli C600 control strain.
Table 3.8 β-lactam MICs of the E. coli C600 OXA-233 pMB3018-transconjugant and E. coli C600. MIC increases relative to the control strain (E. coli C600) are shown in parentheses.
MIC (mg/l)
Antibiotic E. coli C600 E. coli C600/pMB3018
Ampicillin 1.5 >256 (>170×)
Ampicillin-sulbactam 1.5 24 (16×)
Piperacillin 0.75 >256 (>340×)
Piperacillin-tazobactam 0.75 8 (11×)
Amoxicillin 3 >256 (>85×)
Amoxicillin-clavulanate 3 8 (3×)
Temocillin 3 12 (4×)
Cephalotin 3 8 (3×)
Cefuroxime 2 3 (2×)
Cefoxitin 2 4 (2×)
Cefotaxime 0.032 0.064 (2×)
Ceftriaxone 0.047 0.064 (1.4×)
Cefepime 0.016 0.023 (1.4×)
Ceftazidime 0.125 0.19 (1.5×)
Imipenem 0.19 0.19 (1×)
Meropenem 0.012 0.094 (8×)
Doripenem 0.023 0.19 (8×)
Ertapenem 0.004 0.19 (48×)
Aztreonam 0.047 0.064 (1.4×)
Results 72
3.4.3 Impact of KHM-2 on β-lactam resistance
Like for IMP-31 and OXA-233, the blaKHM-2 gene was cloned into the pBK-CMV vector for resistance analysis. As no strain harbouring the reference enzyme KHM-1 was available, the KHM-1 gene was commercially synthesized and also cloned into the pBK-CMV vector, yielding the recombinant plasmid pMB3037. Both plasmids were transformed into E. coli TOP10 and the results of the MIC determination for both strains are shown in Table 3.9. The KHM-2 expressing strain -lactams. The ampicillin MIC was increased moreshowed than very 170 high-fold, MIC while increases expression for someof KHM tested-2 had β only a slight effect on the MIC for piperacillin. The strain was further resistant to amoxicillin with an MIC of >256 mg/l, corresponding to a >256-fold increase compared to the control. The MICs for almost all cephalosporins were >256 mg/l and the highest increases were detected for oxyimino- cephalosporins with more than 8,000-fold for cefotaxime, 5447-fold for ceftriaxone, 1043-fold for cefepime and more than 1024-fold for ceftazidime. Carbapenem MICs were 4 mg/l for
Table 3.9 β-lactam MICs of E. coli TOP10 transformed with the pBK-CMV vector and KHM-2/KHM-1 expressing E. coli TOP10. MIC increases relative to the control strain (E. coli TOP10/pBK-CMV) are shown in parentheses.
MIC (mg/l) E. coli TOP10/pMB3014 E. coli TOP10/pMB3037 Antibiotic E. coli TOP10/pBK-CMV KHM-2 KHM-1
Ampicillin 1.5 >256 (>170×) 64 (43×)
Ampicillin-sulbactam 1.0 96 (96×) 48 (48×)
Piperacillin 1.0 3 (3×) 6 (6×)
Piperacillin-tazobactam 0.75 3 (4×) 6 (5×)
Amoxicillin 2 >256 (>128×) >256 (>128×)
Amoxicillin-clavulanate 1.5 48 (32×) 64 (43×)
Temocillin 8 1024 (128×) >1024 (>128×)
Cephalotin 3 >256 (>85×) >256 (>85×)
Cefuroxime 2 >256 (>128×) >256 (>128×)
Cefoxitin 4 >256 (>64×) >256 (>64×)
Cefotaxime 0.032 >256 (>8,000×) >256 (>8,000×)
Ceftriaxone 0.047 256 (5447×) >256 (>5,447×)
Cefepime 0.023 24 (1043×) 32 (>1,391×)
Ceftazidime 0.25 >256 (>1024×) >256 (>1,024×)
Imipenem 0.19 4 (21×) 2 (10.5×)
Meropenem 0.016 3 (188×) 6 (375×)
Doripenem 0.016 8 (500×) 32 (2,000×)
Ertapenem 0.006 2 (333×) 5 (1,000×)
Aztreonam 0.094 0,094 (1×) 0.094 (1×) Results 73
imipenem, 3 mg/l for meropenem, 8 mg/l for doripenem and 2 mg/l for ertapenem, corresponding to a 21-fold, 188-fold, 500-fold and 333-fold increase compared to the control strain, respectively. The strain expressing the reference enzyme KHM-1 showed lower MIC for ampicillin and ampicillin/sulbactam, while the MIC increases for penicillin, amoxicillin and cephalosporins were mostly the same or equal. Regarding carbapenems, production of KHM-1 led to MICs of 2 mg/l for imipenem, 6 mg/l for meropenem, 32 mg/l for doripenem and 5 mg/l for ertapenem. In comparison to the KHM-2 strain, resistance to imipenem was decreased, but significantly higher for doripenem with a four-times higher MIC. Like for IMP-31 and IMP-1, the expression of KHM-2 and KHM-1 showed no effect on the MIC for aztreonam.
3.4.4 Comparison of IMP-31, OXA-233 and KHM-2 A comparison of the relative MIC increases conferred by production of the three novel carbapenemases identified in this study is shown in Table 3.10. Compared to each other, the
Table 3.10 Relative MIC increases of E. coli TOP10 producing IMP-31, OXA-233 and KHM-2. Shown are the MIC increases in relation to the control strain E. coli TOP10/pBK-CMV. The data are taken from Table 3.6, Table 3.7 and Table 3.8.
MIC increases relative to the control strain E. coli TOP10/pBK-CMV E. coli TOP10/pMB3007 E. coli TOP10/pMB3006 E. coli TOP10/pMB3014 Antibiotic IMP-31 OXA-233 KHM-2
Ampicillin 11× >170× >170×
Ampicillin-sulbactam 12× 12× 96×
Piperacillin 3× 16× 3×
Piperacillin-tazobactam 4× 3× 4×
Amoxicillin 128× >128× >128×
Amoxicillin-clavulanate 11× 4× 32×
Temocillin 2× 1× 128×
Cephalotin 21× 1× >85×
Cefuroxime 32× 1× >128×
Cefoxitin >64× 1× >64×
Cefotaxime 94× 1.5× >8,000×
Ceftriaxone 64× 1× 5447×
Cefepime 32× 1× 1043×
Ceftazidime 128× 1× >1024×
Imipenem 2× 1.3× 21×
Meropenem 12× 2× 188×
Doripenem 12× 4× 500×
Ertapenem 32× 16× 333×
Aztreonam 1× >170× 1× Results 74
enzymes showed distinct differences in thei -lactam resistance. Regarding penicillins, production of both OXA-233 and rKHM impact-2 led on to β significantly higher MICs for ampicillin than production of IMP-31. Both MBL expressing strains showed only slightly elevated MICs for piperacillin and piperacillin/tazobactam. The resistance of the OXA-233 strain towards penicillins was clearly affected by all inhibitors, while the MBL strains were only affected by clavulanic acid. The highest cephalosporin MIC increases were detected for the KHM-2 strain, while the IMP-31 strain was also resistant but with lower total values. In contrast, the production of OXA-233 had nearly no effect on most cephalosporin MICs. The weakest MIC increases for carbapenems were also detected for the OXA-233 strain, as the IMP-31 strain showed increases in resistance of 12 to 32-fold. The KHM-2 strain however showed the highest detected carbapenem MIC increases with up to 500-fold increased resistance. In conclusion, production of KHM-2 led to significantly higher -lactam MICs than production of IMP-31, while the OXA-233 strain overall showed the lowestβ MIC increases. Despite their differences, production of all three enzymes led to increased MICs for carbapenems.
3.5 Purification of IMP-31, OXA-233 and KHM-2 In order to characterize the three novel carbapenemases IMP-31, OXA-233 and KHM-2 biochemically by determining the kinetic parameters Km and kcat in in vitro hydrolysis assays, the enzymes and their respective reference enzymes (IMP-1, OXA-10 and KHM-2) had to be purified at a high level. As overexpression experiments with His- -lactamases did yield very low amounts of purified protein in other studies (Meining, 2012tagged; Hoffmann, β 2013; Lange, 2014), the decision was made to purify the unmodified, native enzymes from larger culture volumes. To acquire a satisfying amount of purified protein, cell extracts from a four-liter E. coli TOP10 culture that harboured one of the respective plasmids pMB3007 (IMP-31), pMB3010 (IMP-1), pMB3006 (OXA-233), pMB3026 (OXA-10), pMB3014 (KHM-2) or pMB3037 (KHM-1) were subjected to two chromatography steps. The first chromatography step for purification was an ion exchange step, separating the respective expressed -lactamase from other proteins with a different isoelectric point (pI). As the resulting enzymeβ preperations still contained contaminating proteins that had the same or similar pI, a gel filtration chromatography was performed as the second purification step. The ion exchange fractions subjected to gel filtration were chosen on the basis of nitrocefin hydrolysis. Nitrocefin is a chromogenic cephalosporin that changes its color -lactamase activity in proteinfrom fractions. yellow Typicalto red when chromatogram hydrolyzed,s fromenabling the anion easy exchange detection and of gel β filtration steps of the purification of KHM-2 are shown in Figure 3.14. Exemplary chromatograms for the other five purified enzymes are shown in the appendix section of this study. In the ion exchange chromatography of cell extracts from the KHM-2 producing E. coli TOP10 strain, the extract was Results 75
applied to the column and high amounts of protein with up to 3,000 milli absorbance units (mAU) were detected during the process. After washing, elution was started by increasing the NaCl concentration with a linear gradient, resulting in changes of the surface charges and elution of the bound protein. The proteins eluted in a single peak that corresponded to an absorbance signal of 2,000 mAU. The corresponding fractions were analyzed for nitrocefin hydrolysis and the ones -lactamase activity were pooled and subjected to gel filtration chromatography.containing In thethis highest second β purification step the proteins were seperated by size and a
Figure 3.14 Ion exchange (A) and gel filtration (B) chromatograms of the KHM-2 FPLC. Shown are the chromogram curves monitored during the chromatography process. The curves show the amount of detected milli absorbance units (mAU) relative to the volume passed through the columns. Red lines indicate the collected fractions. The ion exchange step for KHM-2 was performed at a pH of 7.5. Results 76
single peak was detected after a volume of 67 ml with an absorbance signal of 683 mAU. The corresponding fractions containing high amount of protein were again analyzed for nitrocefin hydrolysis. Fractions showing high activity were pooled and subjected to SDS-PAGE analysis. The results of these analyses are shown in Figure 3.15 for all six enzymes purified in this study. SDS-PAGE analysis showed that all six enzymes were successfully purified with high purity grades and the relative quantity was determined using the GelDox Xr+ software (Bio-Rad). IMP-31 was detected as a band corresponding to a molecular weight of approximately 25 kDa. A second band was detected at a lower weight, but had a clearly lower intensity and relative quantification showed that the IMP-31 enzyme preparation was 85 % pure. IMP-1 was purified near homogenity and was detected as a single band at a weight of approximately 25 kDa with a
Figure 3.15 SDS-PAGE analysis of enzyme preparations of IMP-31, IMP-1, OXA-233, OXA-10, KHM-2 and KHM-1. IMP-31 (A), IMP-1 (B), OXA-233 (C), OXA-10 (D), KHM-2 (E) and KHM-1 (F) were purified by ion exchange and gel filtration chromatography and 10 µl of the preparations were subjected to SDS-PAGE. Shown are images of the Coomassie-stained polyacrylamide gels after electrophoresis. The molecular weights of the size marker bands are stated in kDa. Results 77
purity of 96 %. As the calculated molecular weight for IMP-31 and IMP-1 was 25.116 and 25.113 kDa, respectively, SDS-PAGE analysis showed that the purified proteins had the correct weight. OXA-233 had a calculated weight of 27.571 kDa and was detected as a distinct band at a weight of approximately 25 kDa, while the gel also exhibited a few other significantly smaller bands of contaminating proteins. The calculated purity of the enzyme was 90 %. OXA-10 was also purified near homogenity with a purity of 99 % and was shown as a single distinct band at a weight of approximately 25 kDa, while the calculated weight was 27.550 kDa. SDS-PAGE analysis of the KHM-2 enzyme preparation exhibited a clear band at a weight of 25 kDa. The preparation was slightly contaminated by other proteins that showed weak bands and one protein with a distinct band at a weight corresponding to 55 kDa. However, the calculated purity of the enzyme preparation was 86 %. Like IMP-1 and OXA-10, KHM-1 was purified near homogenity with a purity of 99 % and was detected as a single distinct band in SDS-PAGE at 25 kDa. To acquire a sufficient amount of protein for in vitro hydrolysis assays, all six enzymes were purified in triplicate. Determination of the protein concentration of the enzyme preparations showed that more than 1 mg of purified enzyme was obtained for each preparation of IMP-31, IMP-1, OXA-233, OXA-10, KHM-2 and KHM-1.
3.6 Determination of kinetic parameters -lactams absorb light at ultraviolet wavelengths, it is possible to monitor the enzymatic hydrolysisAs β of these antibiotics spectrophotometrically and to perform Michaelis-Menten kinetics with the obtained data. To analyze the biochemical characteristics of the novel carbapenemases IMP-31, OXA-233 and KHM-2, the purified enzymes and their respective reference enzymes were subjected to determination of the kinetic parameters Km and kcat. Km is the Michaelis constant and an inverse indicator of the affinity of the substrate to the enzyme, while kcat is the turnover number and specifies the amount of substrate molecules that are converted to product per second. The quotient kcat/Km finally is an indicator for the hydrolytic efficiency of the enzyme. By monitoring the absorbance changes in in vitro hydrolysis assays, these kinetic parameters -lactam antibiotics using nonlinear regressionwere determined. An example for of allan absorbance six enzymes curve and and various a Michaelis β -Menten plot is shown in Figure 3.16.
3.6.1 Determination of kinetic parameters for IMP-31 The kinetic data obtained for IMP-31 and IMP-1 are summarized in Table 3.11 and showed that IMP-31 and IMP-1 significantly differed in their hydrolytic activity. Regarding penicillins, IMP-31
-1 -1 -1 showed kcat values of 81 s for penicillin G, 6.6 s for ampicillin and 0.6 s for piperacillin, while
Results 78
Figure 3.16 Hydrolysis assay of IMP-31 for imipenem and Michaelis-Menten plot. (A) IMP-31 hydrolysis curve of 40 µM imipenem. The absorbance was measured at a wavelength of 300 nm. Hydrolysis was initiated by the addition of purified IMP-31 enzyme to a total concentration of 0.02 µM and monitored for 520 seconds. The initial rate velocity was calculated from the linear phase of the reaction which is indicated by a regression line. (B) Imipenem Michaelis- Menten plot for IMP-31. Initial rate velocities (V0) were determined in triplicate for 14 different imipenem concentrations and plotted against these concentrations. Vmax and Km were determined with non-linear regression.
IMP-1 exhibited values of 449 s-1, 81 s-1 and 30 s-1, respectively. In contrast, IMP-31 had clearly lower Km values for penicillin G and piperacillin, resulting in a higher affinity to these substrates.
Although the affinity was higher except for ampicillin, the low kcat values of IMP-31 for penicillins resulted in lower hydrolytic efficiencies than detected for IMP-1 with values of 0.8 µM-1 s-1 for penicillin G, 0.03 µM-1 s-1 for ampicillin and only 0.003 µM-1 s-1 for piperacillin.
The affinity∙ of IMP-31 towards cephalosporins∙ was comparable to IMP-1 with∙ values of 9.4 µM, 41 µM and 13 µM for cefoxitin, ceftazidime and cefotaxime, respectively (IMP-1: 9.4 µM, 41 µM and 2.6 µM). The kcat values for cephalosporins however were lower compared to IMP-1, resulting in clearly lower hydrolytic efficiencies of 1.2 µM-1 s-1 for cefoxitin, 0.6 µM-1 s-1 for
-1 -1 -1 -1 ceftazidime and 2.8 µM s for cefotaxime (IMP-1: 4.4, 9.2 and∙ 7.7 µM s ). IMP-31 was∙ able -1 -1 -1 -1 to hydrolyze carbapenems∙ with hydrolytic efficiencies of 0.5 µM s for imipenem,∙ 1.2 µM s -1 -1 for meropenem and 1.5 µM s for ertapenem. The rates were∙ significantly lower than ∙for -1 -1 -1 -1 -1 -1 IMP-1, which exhibited values∙ of 8.8 µM s , 18 µM s and 5.9 µM s for the three tested carbapenems, respectively. While the IMP∙ -31 Km ∙values for carbapenems∙ were mostly comparable to IMP-1, IMP-31 showed clearly decreased turnover numbers with values of 15 s-1 for imipenem, 2.4 s-1 for meropenem and 4.5 s-1 for ertapenem (IMP-1: 192 s-1, 41 s-1 and 49 s-1), resulting in lowered efficiencies. Both IMP-31 and IMP-1 were not able to hydrolyze the monobactam aztreonam. In conclusion, IMP-31 showed generally lower catalytic efficiencies compared to IMP-1. Although the affinity towards some substrates was higher, the low turnover numbers led to decreased hydrolysis rates for all tested substrates. However, IMP-31 was able to hydrolyze carbapenems and it was finally confirmed that the enzyme has a carbapenemase activity.
Results 79
Table 3.11 Kinetic parameters of IMP-31. The parameters were determined using nonlinear regression and the Michaelis-Menten equitation. Parameters for IMP-1 were determined to serve as a control and reference. Km values are shown in µM and kcat values in s-1. The experiments were performed at 25 °C.
IMP-31 IMP-1
kcat/Km kcat/Km -1 -1 -1 -1 -1 -1 Substrate kcat (s )a Km (µM)a (µM s ) kcat (s ) Km (µM) (µM s ) Penicillin G 81 ± 4.1 108 ± 31 0.8 449 ± 34 256 ± 30 1.8 ∙ ∙ Ampicillin 6.6 ± 0.5 255 ± 23 0.03 81 ± 10 192 ± 51 0.4 Piperacillin 0.6 ± 0.09 185 ± 27 0.003 30 ± 2.3 684 ± 106 0.04 Cefoxitin 11 ± 0.8 9.4 ± 1.6 1.2 41 ± 0.3 9.4 ± 1.2 4.4 Ceftazidime 24 ± 1.2 41 ± 8.1 0.6 377 ± 0.9 41 ± 4.8 9.2 Cefotaxime 36 ± 4.1 13 ± 2.3 2.8 20 ± 0.4 2.6 ± 0.2 7.7 Imipenem 15 ± 1.5 30 ± 3.1 0.5 192 ± 26 22 ± 4.4 8.8 Meropenem 2.4 ± 1.0 2.0 ± 0.7 1.2 41 ± 7.2 2.3 ± 0.7 18 Ertapenem 4.5 ± 0.5 3.0 ± 0.4 1.5 49 ± 4.3 8.3 ± 2.1 5.9 Aztreonam NHb NH - NH NH -
a kcat and Km values represent the means of three independent experiments with three different enzyme preparations ± standard deviations. b NH, no hydrolysis was detected with a substrate concentration of up to 1 mM and an enzyme concentration of up to 200 nM.
3.6.2 Determination of kinetic parameters for OXA-233 -lactamases is carboxylated in vivo, theAs it determination has been shown of kinetic that the parameters active site for serine OXA of-233 class and D OXAβ -10 was performed with sodium bicarbonate as a CO2 source in the buffer used for hydrolysis assays. The kinetic data obtained from these experiments are shown in Table 3.12. OXA-233 exhibited clearly lower turnover numbers for penicillins than OXA-10, with kcat values of 39 s-1 for penicillin G, 343 s-1 and 117 s-1 for oxacillin, for which the kinetic parameters were determined instead of piperacillin due to the presence of an OXA-type enzyme. As OXA-10 exhibited kcat values of 144 s-1 for penicillin G,
690 s-1 for ampicillin and 357 s-1 for oxacillin and as OXA-233 showed higher Km values, the novel enzyme exhibited a lower hydrolytic efficiency for these substrates. Regarding cephalosporins, the hydrolysis rates of OXA-233 for cefoxitin and ceftazidime were extremely low. Although hydrolysis was detectable, the rate was too low to determine the kinetic parameters, as even with extremely high enzyme concentrations of up to 200 nM, the initial rate was not determinable from the monitored absorbance curves. OXA-10 on the other hand was able to hydrolyze these cephalosporins with a low, but determinable rate of 0.003 µM-1 s-1. For cefotaxime, OXA-233 showed a very low hydrolytic efficiency with a Km/kcat ratio ∙of only 0.003 µM-1 s-1, while OXA-10 exhibited an over 10-fold increased value of 0.035 µM-1 s-1 for this substrate. OXA∙ -233 was able to hydrolyze carbapenems with hydrolysis rates of 0.075,∙ 0.2 and
Results 80
Table 3.12 Kinetic parameters of OXA-233. The parameters were determined using nonlinear regression and the Michaelis-Menten equitation. Parameters for OXA-10 were determined to serve as a control and reference. Km values are shown in µM and kcat values in s-1. The experiments were performed at 25 °C.
OXA-233 OXA-10
kcat/Km kcat/Km -1 -1 -1 -1 -1 -1 Substrate kcat (s )a Km (µM)a (µM s ) kcat (s ) Km (µM) (µM s ) Penicillin G 39 ± 1.6 25 ± 5.0 1.6 144 ± 5.1 20 ± 4.0 7.2 ∙ ∙ Ampicillin 343 ± 47 703 ± 8.2 0.5 690 ± 81 444 ± 103 1.6 Oxacillin 117 ± 9.0 470 ± 116 0.25 357 ± 22 148 ± 23 2.4 Cefoxitin NDb ND - 0.2 ± 0.02 65 ± 5.8 0.003 Ceftazidime ND ND - 0.5 ± 0.1 154 ± 25 0.003 Cefotaxime 0.09 ± 0.007 30 ± 2.6 0.003 3.0 ± 0.2 85 ± 6.1 0.035 Imipenem 0.06 ± 0.005 0.8 ± 0.4 0.075 0.16 ± 0.01 0.6 ± 0.2 0.27 Meropenem 0.10 ± 0.005 0.5 ± 0.1 0.2 0.04 ± 0.002 0.3 ± 0.04 0.13 Ertapenem 0.10 ± 0.005 0.8 ± 0.05 0.125 0.06 ± 0.002 0.4 ± 0.1 0.15 Aztreonam NHc NH - 1.2 ± 0.1 192 ± 27 0.006
a kcat and Km values represent the means of three independent experiments with three different enzyme preparations ± standard deviations. b ND, not determinable. Hydrolysis was detectable, but with extremely low rates, preventing determination of kinetic parameters. c NH, no hydrolysis was detected with a substrate concentration of up to 1 mM and an enzyme concentration of up to 200 nM.
0.125 µM-1 s-1 for imipenem, meropenem and ertapenem, respectively. Surprisingly, OXA-10
-1 -1 also showed∙ carbapenem hydrolysis, with hydrolysis rates of 0.27 µM s for imipenem, -1 -1 -1 -1 0.13 µM s for meropenem and 0.15 µM s for ertapenem. OXA-233 ∙therewith showed weaker carbapenem∙ hydrolysis except for meropenem.∙ Both OXA-233 and OXA-10 showed very
Figure 3.17 CO2-dependent imipenem hydrolysis of OXA-233. (A) Absorbance curve for imipenem in phosphate buffer without a CO2 source. The absorbance was measured at a wavelength of 300 nm. Purified OXA-233 enzyme was added to a total concentration of 0.2 µM and the absorbance was monitored for 600 s. (B) Absorbance curve for imipenem in phosphate buffer supplemented with NaHCO3. The experiment was performed equivalent to (A). Results 81
low Km values for carbapenems, ranging from 0.3 to 0.8 µM. This implied a high affinity to these substrates, although the turnover numbers were rather low with values ranging from 0.04 per second to 0.16 per second. In contrast to OXA-10, OXA-233 was not able to hydrolyze aztreonam.
In experiments that were performed without a CO2 source in the reaction mixture, hydrolytic activity against carbapenems was no longer detectable (Figure 3.17). In conclusion and compared to OXA-10, OXA- -lactams except meropenem, resulting233 from showed lower lower kcat values hydrolytic or lower efficiencies substrate for allaffinities, tested while β the lowest rates were detected for cephalosporins.
3.6.3 Determination of kinetic parameters for KHM-2 The kinetic data obtained for KHM-2 and the reference enzyme KHM-1 are shown in Table 3.13. Regarding penicillins, KHM-2 showed significantly higher turnover numbers for penicillin G and ampicillin than KHM-1, with values of 2,101 s-1 and 385 s-1, respectively (KHM-1: 537 s-1 and
198 s-1). However, as the Km value of KHM-2 for penicillin G was more than two times higher than for KHM-1, the higher turnover numbers resulted in a only slightly elevated hydrolytic efficiency compared to KHM-1 with values of 1.8 µM-1 s-1 for KHM-2 and 1.2 µM-1 s-1 for
KHM-1. Both KHM-2 and KHM-1 were able to hydrolyze ∙piperacillin, but with extremely∙ low affinities, as KHM-2 showed a Km value of 3,072 µM for this substrate. This resulted in very low
Table 3.13 Kinetic parameters of KHM-2. The parameters were determined using nonlinear regression and the Michaelis-Menten equitation. Parameters for KHM-1 were determined to serve as a control and reference. Km values are shown in µM and kcat values in s-1. The experiments were performed at 25 °C.
KHM-2 KHM-1
kcat/Km kcat/Km -1 -1 -1 -1 -1 -1 Substrate kcat (s )a Km (µM)a (µM s ) kcat (s ) Km (µM) (µM s ) 2,101 ± 133 1,167 ± 222 1.8 537 ± 63 443 ± 164 1.2 Penicillin G ∙ ∙ Ampicillin 385 ± 27 683 ± 98 0.6 198 ± 23 1,064 ± 174 0.2 Piperacillin 9.9 ± 2.2 3,072 ± 889 0.003 18 ± 2.1 1,136 ± 160 0.0016 Cefoxitin 93 ± 3.9 9.8 ± 0.7 9.5 81 ± 8.5 7.7 ± 1.9 10.5 Ceftazidime 221 ± 26 51 ± 3.2 4.3 105 ± 14 66 ± 9.6 1.6 Cefotaxime 8.1 ± 1.0 5.6 ± 2.6 1.5 64 ± 17 6.0 ± 1.5 11 Imipenem 264 ± 26 52 ± 4.2 5.1 173 ± 49 66 ± 16 2.6 Meropenem 2.6 ± 0.4 3.7 ± 0.3 0.7 1.6 ± 0.2 1.2 ± 0.4 1.3 Ertapenem 2.9 ± 0.1 4.1 ± 0.5 0.7 1.8 ± 0.1 1.4 ± 0.2 1.3 b Aztreonam NH NH - NH NH -
a kcat and Km values represent the means of three independent experiments with three different enzyme preparations ± standard deviations. b NH, no hydrolysis was detected with a substrate concentration of up to 1 mM and an enzyme concentration of up to 200 nM. Results 82
hydrolytic efficiencies of 0.003 µM-1 s-1 and 0.0016 µM-1 s-1 for KHM-2 and KHM-1, respectively. Both enzymes were able to ∙hydrolyze cefoxitin and∙ ceftazidime with similar kcat and Km values, resulting in similar efficiencies. For cefotaxim, KHM-2 however showed a significantly decreased activity, as the turnover number was eight times lower than for KHM-1 with a value of 8.1 s-1, while KHM-1 exhibited a value of 64 s-1. This resulted in a hydrolytic efficiency of only 1.5 µM-1 s-1, while KHM-1 was able to hydrolyze this substrate with a rate of 11 µM-1 s-1.
Regarding∙ carbapenems, KHM-2 and KHM-1 clearly differed in their ability to hydrolyze∙ imipenem. KHM-2 exhibited a kcat value of 264 s-1 and a Km value of 52 µM, while KHM-1 showed a turnover number of only 173 s-1 and a similar Km value of 66 µM, resulting in a two times higher efficiency of KHM-2. Meropenem and ertapenem on the other hand were hydrolyzed with similar rates of 0.7 µM-1 s-1 for KHM-2 and 1.3 µM-1 s-1 for KHM-1, as the kcat and Km values were also relatively similar.∙ Like the other metallo-∙ -lactamases characterized in this study, both KHM-2 and KHM-1 were not able to hydrolyze theβ monobactam aztreonam. In conclusion, KHM-2 showed a more efficient hydrolysis of penicillins than KHM-1, while cephalosporin hydrolysis was similar except for cefotaxime. Both enzymes hydrolyzed carbapenems with KHM-2 showing a two times higher hydrolytic efficiency against imipenem.
3.6.4 Comparison of the hydrolytic efficiencies of IMP-31, OXA-233 and KHM-2 The kinetic parameters of the three novel carbapenemases identified and characterized in this study are listed comparatively in Table 3.14. Compared to each other, KHM-2 showed the highest penicillin hydrolysis rates. While the rate for penicillin G was comparable to OXA-233 with values of 1.8 µM-1 s-1 and 1.6 µM-1 s-1, respectively, IMP-31 hydrolyzed this substrate with
-1 -1 a rate of 0.8 µM s ∙ and furthermore∙ showed a significantly lower rate for ampicillin -1 -1 -1 -1 (0.03 µM s ) than∙ OXA-233 and KHM-1 (0.5 and 0.6 µM s ). Regarding cephalosporins, KHM-2 showed∙ the highest rates for cefoxitin and ceftazidime,∙ while IMP-31 exhibited clearly lower rates. OXA-233 was able to hydrolyze these antibiotics, but with extremely low hydrolyis rates, preventing a determination of the kcat and Km parameters. For cefotaxime, hydrolysis rates were determinable for all three enzymes and IMP-31 showed the highest rate with 2.8 µM-1 s-1,
-1 -1 while the rate for OXA-233 was again very low with 0.003 µM s . The hydrolytic activity∙ of IMP-31, OXA-233 and KHM-2 against carbapenem antibiotics showed∙ that OXA-233 was a rather weak carbapenemase with rates of only 0.075 µM-1 s-1 for imipenem, 0.2 µM-1 s-1 for
-1 -1 meropenem and 0.125 µM s for ertapenem. Although∙ IMP-31 also showed a relatively∙ low -1 -1 hydrolysis rate for imipenem∙ (0.5 µM s ), the enzyme exhibited the highest detected rates for -1 -1 -1 -1 meropenem and ertapenem with values∙ of 1.2 µM s and 1.5 µM s , respectively. Imipenem -1 -1 on the other hand was most efficiently hydrolyzed ∙by KHM-2 with a∙ rate of 5.1 µM s , while -1 -1 the enzyme showed rates of 0.7 µM s for both meropenem and ertapenem. In conclusion,∙ the ∙ Results 83
Table 3.14 Comparison of the hydrolytic efficiencies of IMP-31, OXA-233 and KHM-2. The data are taken from Table 3.11, Table 3.12 and Table 3.13. The data are based on hydrolysis experiments performed at 25 °C.
kcat/Km (µM-1 s-1)a Substrate IMP-31 OXA-233 KHM-2 ∙ Penicillin G 0.8 1.6 1.8 Ampicillin 0.03 0.5 0.6 Oxacillin NAb 0.25 NA Piperacillin 0.003 NA 0.003 Cefoxitin 1.2 NDc 9.5 Ceftazidime 0.6 ND 4.3 Cefotaxime 2.8 0.003 1.5 Imipenem 0.5 0.075 5.1 Meropenem 1.2 0.2 0.7 Ertapenem 1.5 0.125 0.7 Aztreonam - d - - a The data are based on kcat and Km means of three independent experiments with three different enzyme preparations. b NA, Hydrolysis was not analyzed for these enzyme/substrate combinations. c ND, Hydrolysis was detectable, but too low to determine the kinetic parameters. d No hydrolysis was detectable for aztreonam. two metallo- -lactamases exhibited clearly higher carbapenem hydrolysis rates than the class D enzyme OXAβ-233. Despite the difference between the three enzymes, the hydrolysis assays -lactamases identified in this study were carbapenemases. clearly showed that all three novel β 3.7 Determination of the isoelectric point of IMP-31, OXA-233 and KHM-2 -lactamases were classically identified andPrior subdivided to the wide by availability isoelectric of focussing sequencing (IEF) techniques, and determination β of their isoelectric point. By incubation of the IEF -lactamase bands can easily be visualized based upon the hydrolysis of nitrocefingel with which nitrocefin, leads to β a color change from yellow to red. Nowadays, the pI is -lactamases and is mostly determined in silico. noHowever, longer the used calculated as a separation pI can still marker differ forsignificantly β from the experimental pI and isoelectric focussing is still a useful tool for comparison of strains that express the same -lactamase or to
-lactamase. β distinguishFor determination them from of strainsthe pI thatof the produce three a noveldifferent car βbapenemases in this study, the purified enzymes were subjected to isoelectric focussing, followed by nitrocefin analysis and the results obtained from these experiments are shown in Figure 3.18. For both OXA-233 and OXA-10, a single signal was detected at a pI of approximately 6.7, while the calculated pI for both enzymes was 6.96. The calculated pI for IMP-31 was 8.46; however the enzyme was detected with a single
Results 84
Figure 3.18 Isoelectric focussing of OXA-233, OXA-10, IMP-31, IMP-1, KHM-2 and KHM-1. FPLC-purified - lactamases TEM-1, TEM-3, SHV-3, SHV-1, SHV-5 and CMY served as a pI marker. enzymes were used for IEF. Shown are the IEF gels after incubation with nitrocefin. A standard containing the β signal at a pI of approximately 9.1 in IEF. IMP-1 was detected at a pI of approximately 8.0, also differing from the calculated pI of 8.46. Both KHM-2 and KHM-1 exhibited lower pIs than IMP-31 and IMP-1 and were detected at a height of 6.9 for KHM-2 and 7.6 for KHM-1, also showing differences to the calculated pIs of 6.26 for KHM-2 and 7.18 for KHM-1. In each of the KHM-2 and KHM-1 lanes, an unspecific signal with a clearly lower intensity was detected, most likely resulting from degraded KHM enzymes, which is a common and frequently observed IEF phenomenon.
3.8 Sequencing and characterization of the blaOXA-233 carrying plasmid pMB3018
The blaOXA-233-carrying wildtype plasmid pMB3018 was identified by transconjugation experiments and nuclease S1-Southern blots in this study. To acquire more information on pMB3018, the plasmid was isolated from a culture of the E. coli C600 transconjugant and fully sequenced using the 454-pyrosequencing technique. By assembling the sequence reads, a single contig with a size of 52,278 bp was obtained, covered by 86,248 single sequence reads. Sequence analysis revealed that the contig was circularizable and that it covered the whole pMB3018 sequence. pMB3018 exhibited a GC content of 48.85 % and sequence analysis showed that the plasmid carried 58 open reading frames, which were annotated with the help of the NCBI nucleotide database. A circular map of the annotated plasmid is shown in Figure 3.19. While most ORFs could be assigned to genes coding for known proteins, several were only putative genes. Most genes identified in the sequence coded for plasmid infrastructure proteins. Two
Results 85
Figure 3.19 Circular map of pMB3018. The outer circle displays the size in bp, the inner circle represents the GC content plotted against the average of the complete sequence with pale green indicating a GC content higher and purple indicating a GC content lower than the average of the total sequence. Genes are color-coded, depending on functional annotations: blue, conjugative transfer; orange, plasmid replication and maintenance; red, antimicrobial resistance; green, gene integration or transposition; and grey, putative functions or hypothetical proteins. large transfer operons were identified, containing the tra genes A, B, C, D, E, F, G, H, I, J, K, L, M and O, the endonuclease encoding gene nuc and the oriT region. The second locus consisted of the tra genes K, J, I and the partially deleted fipA gene. Other genes carried by pMB3018 that play a role in plasmid stability, antirestriction mechanisms, host range determination or regulation of conjugation were stbA, B and C, ardR, B and K and kikA. The oriV region was identified at the positions 17020 to 18035, containing a repA replicase gene. Regarding genes conferring antibiotic resistance, the aac(6’), qacEΔ and sul genes carried by the blaOXA-233 integron were the only ones identified on pMB3018. Beside two genes that code for putative phage integrases, two transposase genes, IS6100 and ISSen4, were identified. A total of 17 ORFs identified in the Results 86
sequence were not annotable as a BLAST search yielded only hits for hypothetical genes without a predicted function. As regions with significant differences in the GC content are often indicators for the insertion of mobile elements, the sequence was analyzed for these regions. The results are visualized in Figure 3.19 as the GC content plot. Two regions were identified that significantly differed from the average. The first region consisted of the two ORFs with unknown function neighbored to the ISSen4 transposase gene at positions 8253 to 11067. This region exhibited a GC content of only 30.6 %, while the average of the plasmid was 48.85 % as noted before. The second region consisted of the blaOXA-233 carrying integron and the neighboring IS6100 transposase gene with a
GC content of 56.6 %. The blaOXA-233 gene however had a GC content of only 42 % and if not taken into consideration, the GC content of the region was 59 % and therewith more than 10 % higher than the average of pMB3018. The sequence was furthermore analyzed for repeat regions as these often serve as markers of a transposon or insertion sequence. Flanking the IS6100 gene, two inverted repeat regions with a length of 123 bp each were identified, indicating the presence of a transposon that consisted of only the transposase gene. Regarding the blaOXA-233-carrying integron, a 13-bp inverted repeat region was identified upstream of the intI gene, with the counterpart located downstream of the IS6100 transposase gene. No other repeat regions were identified in the sequence which were correlatable to a potential insertion sequence or transposon, especially not to the ISSen4 transposase gene. Replicon type analysis revealed that the plasmid had no known replicon type, but was related to the IncN type. A BLAST homology search revealed four plasmids with homologies to pMB3018, however they showed significant differences. The closest relative was the NDM-1 carrying plasmid pJIE137 (accession number NG_037697.1) with a coverage of 71 % and an identity of 95 %. The next relatives with high homology scores were pECS01 (accession number KJ413946.1) and pTR3 (accession number JQ349086.2), both showing a coverage of 62 % and an identity of 97 %. p271A (accession number JF785549.1) finally was the fourth relative with acoverage of 52 % and an identity of 97 %. A schematic comparison of pMB3018 and the four other plasmids is shown in Figure 3.20. Using the progressiveMauve algorithm, ten regions with high homologies were identified in pMB3018 that were also found in one or more of the related plasmids. The regions found in all five plasmids were the two tra regions, including their neighbored genes kikA, ΔfipA and the stbACB genes and the oriV region including the repA and ardK genes. The region containing the genes ardR, ardB, ccgC and mpr were also found in pJIE137, pECS01 and pTR3. In p271A, only a part of this region including the mpr gene was identified. The 6-kb region containing the ISSen4 transposase gene that was part of p271A, pECS01 and pTR3 was shortened in pMB3018, although the ISSen4 gene was still present. The pMB3018 region from 8252 to 16368 bp which contained the two phage integrase genes and the three neighbored putative genes was not found in any of the four related plasmids. Results 87
The alignment was generated using the Comparison of pMB3018, pJIE137, p271A, pECS01 and pTR3.
20 . 3
Figure Figure progressiveMauve algorithm. Regions with high homologies are coloredand similarities are indicated with a homology plot. The annotated genes corresponding the to identified regions ofpMB3018 are shownabove. Results 88
Among the four plasmids related to pMB3018, only pJIE137 exhibited regions with similarities to the resistance gene region of pMB3018. Analysis showed that pJIE137 also carried a class 1 integron and the regions that showed high homologies to pMB3018 were the ones coding for the conserved integron genes intI1, qacEΔ1 and sul1. In addition, pJIE137 showed a homology to the IS6100 transposase gene identified in pMB3018. In conclusion, the differences found in this analysis showed the distinct differences of pMB3018 in comparison to other known IncN-related plasmids. Discussion 89
4 Discussion
The ongoing spread and diversification of carbapenemases in Gram-negative pathogens is one of the most urgent problems for antimicrobial therapy of healthcare-associated infections. As these -lactamases can significantly -lactam antibiotics, theβ identification and characterizationdiffer in theirof these ability enzymes to hydrolyze is crucial different for clinical β diagnostics and correct antimicrobial treatment. In this study, three carbapenem-resistant Gram-negative clinical isolates from patients hospitalized in Germany were analyzed for the presence of a novel carbapenemase.
4.1 Identification of IMP-31 The isolate P. aeruginosa NRZ-00156 showed a carbapenemase phenotype, as the modified Hodge Test indicated secretion of a carbapenemase into the medium, although this has been described as rather based on leakage than secretion (Livermore, 1995). As carbapenem- resistant clinical P. aeruginosa isolates are often carriers of metallo- -lactamases (Walsh, 2010;
Diene & Rolain, 2014), the isolate was analyzed by an EDTA-CDT. Theβ inhibition of carbapenem resistance by EDTA clearly indicated the presence of an MBL, as these enzymes require one or two zinc ions to perform the nucleophil -lactam ring. It has to be noted, that
EDTA itself also has an inhibitory effect icon attack cell growth, on the asβ seen for the blank disk with EDTA. This is also based on the chelating characteristics of EDTA, making free metal ions unavailable for the bacteria (Root et al., 1988). However, the inhibitory effect of EDTA is considerably weaker than growth inhibition by carbapenem antibiotics. -lactam MICs of the isolate were determined and clearly showed increased resistance to
βcarbapenems. Although carbapenem resistance can be based upon other mechanisms, such as loss of the OprD porin, an MIC of >32 mg/l for all tested carbapenems further indicated the presence of a carbapenemase, as mutation or loss of porin very rarely leads to carbapenem MICs higher than 32 mg/l (Livermore, 2001). In contrast to MICs for other penicillins and cephalosporins, the isolate did not show elevated MICs for piperacillin. This has been shown for many metallo- -lactamases, although it has not been described as a typical characteristic (Laraki et al., 1999; Franceschiniβ et al., 2000; Poirel et al., 2000; Cornaglia et al., 2011; Yong et al., 2012). Consequently, the lower piperacillin MIC was only a hint for a potential MBL presence. The susceptibility towards aztreonam finally was another indicator for an MBL production, as these enzymes are not able to hydrolyze this antibiotic (Cornaglia et al., 2011).
The identification of the blaIMP-31 gene in the isolate by PCR and sequencing was surprising, as the same PCR for blaIMP genes was performed prior to this study in the routine diagnostic Discussion 90
process. It remains unclear why the gene was not detected in clinical diagnostics, but the most likely explanation would be an insufficient quality of the DNA used for PCR analysis. IMP-type (IMP for “active on imipenem”) carbapenemases show a continuous worldwide spread among almost all Gram-negative pathogens, but are mostly found in P. aeruginosa and A. baumannii isolates (Zhao & Hu, 2011). The first IMP-type enzyme was identified in 1988 in a
P. aeruginosa strain from Japan (Watanabe et al., 1991) and since then, blaIMP genes have been increasingly detected wordwide (Zhao & Hu, 2011). In Europe, IMP-type carbapenemases have been reported from Austria, Italy, the Czech Republic, France, the UK, Slovakia and Germany (Riccio et al., 2000; Tysall et al., 2002; Neuwirth et al., 2007; Ohlasova et al., 2007; Duljasz et al., 2009; Nemec et al., 2010; Pournaras et al., 2013). To date, 50 unique IMP-type enzymes have been assigned (http://www.lahey.org/studies/), showing a high diversity of their amino acid sequences with up to 22 % differences. At the time of the discovery of IMP-31, the enzyme showed a very high diversity towards all other known IMP-type enzymes with only 84.1 % homology to the next nearest relative IMP-8. The greatest distance was found in comparison to IMP-38 to which IMP-31 showed 54 single amino acid substitutions, resulting in a homology of only 78.2 %, which is currently the highest diversity between any IMP-type enzymes. In addition, IMP-31 was the most distant enzyme compared to the reference enzyme IMP-1 with a homology of only 80.0 %. However, the description of IMP-35 shortly later revealed a closer relative with a homology of 96.7 %. Unlike IMP-35 and most other IMP variants that consist of 246 amino acid residues, IMP-31 was a 245 amino acid protein, shortened by one C-terminal residue. In general, the C-terminus of IMP-31 was significantly different from those of other IMP-type enzymes. For most IMP-type enzymes the C-terminus is formed by a KKPSXPSN motif, with the first two lysine residues being conserved in all known IMP variants. For IMP-31 the C- terminal sequence was KNHHSPK, making the C-terminus of the enzyme the most divergent compared to all other IMP-type metallo- -lactamases. Although relatively highly conserved, the function of the C-terminus of IMP-type enzymesβ is still unknown, so it can only be speculated about the influence of the altered C-terminus. For serine- -lactamases, it is thought that they originate from penicillin-binding-proteins and they share severalβ structure similarities (Kelly et al., 1986). As the C-terminus of PBPs is involved in interaction with the membranes (Harris et al., 2002), the C-terminus of serine- -lactamase could have the same function. However, MBLs represent a completely differentβ class of enzymes, as they do not belong to the SxxK acetyltransferases superfamiliy like serine- -lactamases and are part of their own superfamily of metallo-enzymes (Cornaglia et al., 2011).β Consequently, nothing equivalent is known for the C-terminus of MBLs. As the signal peptide for the periplasmatic localization -lactamases is located at the N-terminus (Pradel et al., 2009), it is furthermore unlikely thatof β the altered C- terminus of IMP-1 has a direct influence on secretion of the enzyme. Discussion 91
The phylogenetic analysis of IMP-31 and the clustering of all IMP-type enzymes into thirteen groups underlined the growing evolutionary complexity of the IMP family. The phylogenetic tree showed IMP-31 to be a member of a new phylogenetic cluster that consists only of IMP-31 and IMP-35 and is the most distant cluster of the IMP family. With a greater evolutionary distance to a putative common ancestor even greater than IMP-35, IMP-31 further illustrated the immense diversity of IMP-type carbapenemases and IMP-31 in particular. As IMP-35 was found in a P. aeruginosa isolate in the Dutch-German border region and as the IMP-31 carrying strain was referred to the National Reference Laboratory for Gram-negative pathogens from a clinical diagnostics lab from North-Rhine-Westphalia, it can be assumed that the IMP-31 cluster has established itself in Western Germany. Furthermore, the isolate P. aeruginosa NRZ-00156 expressed the allelic profile ST235, which has been frequently reported as a carrier of different carbapenemases, e.g. IMP-1, IMP-6, VIM-2, GES-6 or PER-1 (Libisch et al., 2008; Seok et al., 2011; Sardelic et al., 2012; Botelho et al., 2015; Shimizu et al., 2015). ST235 is an international P. aeruginosa high-risk clone which belongs to the clonal complex CC235 (Maatallah et al., 2011). CC235 has been frequently reported in context with the production of several metallo- - lactamases and ESBLs like IMP-7, VIM-4 or GES-1 in European countries (Nemec et al., 2010β; Samuelsen et al., 2010; Larché et al., 2012). The identification of IMP-31 in such a sequence type is a strong indicator for a potential spread of this strain and the IMP-31 MBL in healthcare settings. With a GC content of ~39 %, it is very clear that all IMP variants are introduced into the the species P. aeruginosa, which has a GC content of ~66 %, but the source of these genes is still unknown. It can be assumed that IMP genes originate from intrinsic genes of one or several closely related environmental bacterial species, although these progenitors are hitherto unknown, unlike the progenitors of e.g. CTX-M, OXA-48 or OXA-23 (Poirel et al., 2004a; Poirel et al., 2008; Cantón et al., 2012b). Due to the high diversity of IMP-31 and IMP-35 compared to other variants it can be speculated, that they rather represent a de novo mobilization from the unknown environmental source than an evolutionary diversification from other IMP-type enzymes within P. aeruginosa. This would further imply the danger of the continuing introduction of novel resistance genes into bacterial species of medical importance.
Most metallo- -lactamase genes and all blaIMP genes in particular were found within integron structures (Zhaoβ & Hu, 2011). This was also the case for blaIMP-31. The gene showed a genetic environment very similar to blaIMP-35, which also was the first gene cassette in a class 1 integron, followed by blaOXA-35 and aac(6’)-Ib genes (Pournaras et al., 2013). However, the sequence downstream of the aac(6’)-Ib gene showed major differences to the blaIMP-35-carrying integron with two additional resistance genes, aac(3)-Ic and aphA15, which were cassettes of the integron. A comparison of the genetic environments is shown in Figure 4.1. The blaIMP-31 integron
Discussion 92
Figure 4.1 Comparison of the genetic environment of blaIMP-31 and blaIMP-35. Grey boxes indicate similar sequences. The figure for the blaIMP-35 genetic environment was obtained and modified from Pournaras et al. (2013). was furthermore disrupted by the transposon-associated genes tniC and tnpA, encoding a recombinase of the invertase/resolvase family (Radstrom et al., 1994) and the transposase A.
The disruption of a class 1 integron by the tniC gene has been described for a blaVIM-2-carrying putative transposon that is related to the worldwide dispersed Tn5090/Tn402 transposon, which is proposed to be the progenitor of the common class 1 type of integrons (Radstrom et al., 1994; Toleman et al., 2007; Moyo et al., 2015). Although Tn5090/Tn402 consists of the additional genes orf6, tniB and tniA downstream of the tniC gene, which all code for transposition enzymes, the presence of tniC is sufficient for transposition (Toleman et al., 2007).
This strongly suggests a possible transposon-mediated mobilization of blaIMP-31, as it has also been shown for a Tn5090/Tn402-associated blaVIM-1-carrying integron (Tato et al., 2010). As the sequence obtained by PCRs and genome walking did not cover the whole putative transposon structure, for example the inverted repeat region that would be located upstream of the intI gene, it was however not definitely possible to determine the functionality of the putative transposon. In addition, it is not clear if the transposon-like structure only disrupted the integron or if the conserved 3’CS end has been completely deleted and replaced by the transposon elements, as it has been shown for a VIM-2 carrying class 1 integron (Samuelsen et al., 2009). In contrast, the blaIMP-35-carrying integron was not disrupted by such elements. The differing gene arrangement of the genetic environments of the two MBL genes however is difficult to explain. Integron gene cassettes are always integrated next to the attI site and the gene arrangement correlates with the chronological order of integration. This means that the aphA15 and aac(3)-Ic gene cassettes of the blaIMP-31-carrying integron were the first gene cassettes that were integrated. Subsequently, the aac(6’)-Ib, blaOXA-35 and blaIMP-31 genes must have been integrated in this order and the exact same genetic events must have been occurred in another integron with the only difference beeing the integration of a very closely related blaIMP gene. In conclusion, this implies that it is rather unlikely that IMP-31 and IMP-35 originated directly from each other or that one of the encoding integron-bourne genes simply mutated while integrated. It is more likely that both genes represent two separate integration events; Discussion 93
however the exact same order of the two following genes is still remarkable. In addition, the blaIMP-35-harbouring isolate expressed another sequence type, ST622, although this sequence type is very closely related to ST235 (Pournaras et al., 2013). This further implied that the two genes did not originate directly from each other and possibly represent two separate de novo mobilizations from a still unknown source of IMP-type carbapenemase genes.
Expression of the blaIMP-31 gene cassette and the following cassettes of the disrupted integron was controlled by a typical integron promoter structure. Integron promoters are part of the integrase gene and the attI site and several types of promoters have been identified and classified on the basis of their impact on gene cassette expression. The promoter found in the blaIMP-31-carrying integron was the Pc hybrid 2 promoter (PcH2), consisting of the strong -35 region TTGACA and the weaker -10 box TAAGCT (Papagiannitsis et al., 2009). This combination has been identified as a weaker promoter than the perfect strong integron promoter, which exhibits the -10 box TAAACT instead (Collis & Hall, 1995). It has been shown that expression of genes controlled by the PcH2 variant is reduced almost 4-fold in comparison to the strong variant (Papagiannitsis et al., 2009). The P2 promoter was identified directly adjacent to the intI gene and was missing the insertion of three guanine bases. In the active variant of P2, this insertion leads to an optimization of the spacing between the -35 and -10 boxes to 17 bp. A lack of this insertion leads to an inactivation of the promoter (Collis & Hall, 1995). An active P2 promoter has been described as a compensator for a weak or hybrid Pc promoter (Papagiannitsis et al., 2009), but this was absent in the present integron. Consequently, it can be assumed that the expression of the IMP-31-encoding gene was not at the highest possible level. As the isolate showed high carbapenem resistance that was exclusively reversible by inhibition with EDTA (and thus showed the significant contribution of IMP-31 towards resistance) an expression sufficient to confer high level resistance was indicated. Contrary to many other acquired MBL genes that are plasmid-encoded (Walsh et al., 2005),
Southern blot experiments showed that the blaIMP-31 gene was located on the bacterial chromosome. A number of IMP variants were described as chromosome-encoded, e.g. IMP-24 (Lee et al., 2008), IMP-28 (Perez-Llarena et al., 2012) or IMP-33 (Deshpande et al., 2013). On the other hand, several other enzymes like IMP-29 (Jeannot et al., 2012) or IMP-34 (Shigemoto et al., 2013) have been described as plamid-encoded. As integrons can not mobilize themselves to move to another organism, it must be assumed that the blaIMP-31 gene was mobilized from an unknown source into the P. aeruginosa isolate NRZ-00156. Consequently, a conjugative plasmid or conjugative transposon must have been present for mobilization of blaIMP-31 and latter could still be the case. As gene cassettes in integrons furthermore can easily be excised and integrated into another integron or transposon structure, it is very likely that the blaIMP-31 gene is mobilizable into other organisms.
Discussion 94
4.2 Identification of OXA-233 The isolate C. freundii NRZ-02127 also exhibited a carbapenemase phenotype with a positive modified Hodge Test. However, the resistance spectrum that was detected in MIC analysis did not match any known carbapenemase profile, as the high resistance towards penicillins, the inhibition by clavulanic acid and the susceptibility to cephalosporins did neither fit to class A carbapenemases nor to MBLs or OXA-48. A sparing of oxyimino-cephalosporins is untypical for class A carbapenemases, although they are inhibited by clavulanic acid (Nordmann et al., 2012). MBLs also do usually not spare cephalosporins and are unsusceptible to clavulanic acid (Cornaglia et al., 2011). OXA-48 finally shows lowered hydrolysis of cephalosporins, but is also not inhibited by clavulanic acid (Poirel et al., 2004b). Therefore, the discovery of the OXA-10 related enzyme OXA-233 was surprising, as OXA-10-like enzymes were classified as narrow- or extended-spectrum enzymes without carbapenemase activity (Poirel et al., 2010; Evans & Amyes, 2014). Enzymes of the OXA-10 group were detected first in the U.S. (Korfhagen et al., 1975) and are found worldwide today (Poirel et al., 2010). In Europe, OXA-10 related enzymes have been reported in P. aeruginosa isolates from Turkey (Hall et al., 1993; Danel et al., 1995; Danel et al., 1998; Danel et al., 1999), Germany (Pournaras et al., 2013), Croatia (Sardelic et al., 2012), Bulgaria (Vatcheva-Dobrevska et al., 2013), Portugal (Moura et al., 2012), France (Aubert et al., 2001; Poirel et al., 2001; Fournier et al., 2010a; Hocquet et al., 2011) and Denmark (Hansen et al., 2014). OXA-233 was very closely related to OXA-35 and OXA-10 with one and two amino acid substitutions, respectively. At position 117, OXA-233 possessed a phenylalanine residue, while all other OXA variants exhibit a valine, isoleucine or rarely, leucine (Leonard et al., 2013). As this mutation was the only one that divided OXA-233 and OXA-17 and as both OXA-10 and OXA-17 and most other OXA-10 related enzymes were classified as only narrow- or extended-spectrum enzymes (Poirel et al., 2010), it was assumed that the V177F substitution was responsible for the carbapenemase activity. blaOXA-233 was identified to be part of a conserved and intact class 1 integron, as it has been shown for numerous other OXA-type genes, for example blaOXA-28 (Poirel et al., 2001), blaOXA-35
(Aubert et al., 2001), blaOXA-142 (Liu et al., 2014) or blaOXA-320 (Cicek et al., 2014). The OXA-233 encoding gene was the second gene cassette of the integron. As the expression of gene cassettes within an integron continously decreases with an increasing distance from the promoter (Gillings, 2014), a lower expression than for the adjacent aac(6´)-Ib gene can be assumed. However, as the in silico promoter analysis of the integron revealed that a strong Pc promoter was present, the effect should be minimized as the strong variant of the Pc promoter shows an expression level almost four times higher than the PcH2 variant identified in the blaIMP-31- carrying integron, as previously described. As the clinical isolate furthermore exhibited elevated MICs for penicillins and carbapenems and as OXA- -lactamase
233 seemed to be the only β Discussion 95
present in the isolate, the influence of the position in the integron on expression can be assumed as rather low. Like for the blaIMP-31-carrying integron, the P2 promoter was found in its inactive variant, missing the space-optimizing insertion of three guanines between the -35 and -10 boxes and therewith it can be assumed that P2 had no influence on the expression of blaOXA-233. No transposon structures were identified in the genetic environment of the blaOXA-233 gene and analysis of the full sequence of the plasmid pMB3018 showed that the gene was not obviously part of a transposon, which will be discussed later.
The localization of blaOXA-233 was identified by Southern blot analysis with a blaOXA-233 specific probe, which showed that the blaOXA-233 was located on this transconjugable plasmid (pMB3018) with a size of approximately 50 kb. Nuclease S1 digestion revealed two other plasmid bands of C. freundii NRZ-02127. These bands are likely to represent two other plasmids with a greater size than pMB3018, but it is also possible that they resulted from circularized and supercoiled forms of pMB3018 due to an incomplete digestion by nuclease S1. As the hybridization however showed no signals for blaOXA-233 corresponding to these bands and as the OXA-233 transconjugant did not exhibit these additional bands, an incomplete digestion is very unlikely. The signals that were detected at a size of approximately 680 kb represented unspecific hybridizations with the intense bands observed in PFGE at the same size. This is a common phenomenon in PFGE analyses.
Almost all blaOXA group genes have been found on plasmids, with the exception of the more recently found intrinsic OXA variants in some Acinetobacter species, for example OXA-213, OXA-235 or OXA-309 (Evans & Amyes, 2014). The most prominent and clinically relevant plasmid-encoded CHDL is OXA-48, which can be harboured by the plasmid pOXA-48a and close relatives which have shown an almost worldwide spread (Poirel et al., 2012a). Among the OXA-10 group, many enzymes have been described as plasmid-encoded, for example OXA-7 in E. coli, OXA-10 in P. aeruginosa and OXA-17 also in P. aeruginosa (Philippon et al., 1983;
Medeiros et al., 1985; Danel et al., 1999). The plasmid localization of blaOXA-233 in combination with the presence of a class 1 integron implies a high ability to spread into other organisms. As pMB3018 is at least conjugable from C. freundii into E. coli, it is very likely that it is also transferable into other Enterobacteriaceae species of clinical importance.
4.3 Identification of KHM-2 Like P. aeruginosa NRZ-00156, the isolate P. aeruginosa NRZ-03096 showed a carbapenemase phenotype in the modified Hodge Test. This was further underlined by the performance of the EDTA-CDT which indicated the presence of an MBL. However, the inhibition zone increase for the blank control disk was much greater than for the IMP-31 producing strain with 12 mm for P. aeruginosa NRZ-00156 and 19 mm for P. aeruginosa NRZ-03096. This phenomenon is frequently observed in clinical diagnostics and is based upon the differing susceptibilities of the Discussion 96
different tested isolates to EDTA (Pitout et al., 2005; Galani et al., 2008). Like the IMP-31 isolate, P. aeruginosa NRZ-03096 showed low MICs towards piperacillin, further indicating an MBL production, as simultaneously MICs for other penicillins, cephalosporins and carbapenems were elevated. Carbapenem MICs however were only slightly elevated and the isolate was susceptible to imipenem and only indermediate resistant to meropenem according to the EUCAST criteria, which would have been unusal in case of an MBL production. Shotgun cloning experiments however revealed that P. aeruginosa NRZ-03096 harboured a novel metallo- -lactamase which had a homology of only 74.3 % to the KHM-1 enzyme (KHM for
“Kyorin Healthβ Science MBL1”), which has been first described in 2008 and was found only once in a clinical C. freundii isolate from Japan (Sekiguchi et al., 2008). As the homology to the next -lactamases of the same type, it was considered that relativethe novel was enzyme very lowcould compared represent to a othernovel βenzyme type, but as the threshold for the definition of a new type is proposed at <73 % homology (George Jacoby, personal communication), the enzyme was designated as KHM-2. The mutations of KHM-2 in comparison to KHM-1 were spread widely over the whole enzyme, but the highly conserved zinc-binding motifs of subclass B1 enzymes were not altered. As stated in the results section, this applied only to the histidine residues directly involved in zinc-binding, as the sequence exhibited a threonine to aspartic acid substitution at position 100, which is part of the conserved HXHXD zinc binding motif. A potential influence on the zinc binding efficiency will be discussed later.
Regarding the genetic environment of the blaKHM-2 gene, it was surprising that no integron structures were found, as most other MBL genes were described as integron-bourne (Cornaglia et al., 2011). The identification of two putative transposase genes adjacent to the blaKHM-2 gene however, strongly suggests a mobility of the gene, although no repeat regions could be idenified as associated with the putative transposon structure. The insE transposase gene has been described as part of a large insertion sequence flanked by two IS903 elements, which are members of the IS5 family (Sekizuka et al., 2011). In IS903, insE was furthermore identified as associated with the chaperon-enconding genes groEL and groES upstream of the 5´ end of insE.
An association of insE with these genes was also identified in a blaNDM-1-carrying transposon from A. baumannii, while insE was also not associated with repeat regions in this context (Pfeifer et al., 2011), as it was detected for blaKHM-2. As the sequence of the genetic environment of blaKHM-2 obtained by shotgun cloning did not provide information on the genes located further downstream of the insE gene, a possible association with the chaperon-encoding genes groEL and groES could not be analyzed. Regarding the gene coding for the putative transposase of the ISXo2 family, it was not possible to identify any putative repeat region adjacent to the ORF, especially as the obtained sequence from shotgun cloning did not cover the whole ORF. ISXo2 has been described as an insertion sequence found in Xanthomonas oryzae pv. oryzae (Rajeshwari & Sonti, 2000) but has not yet been described as beeing associated with any type of Discussion 97
antibiotic resistance. However, a BLAST homology search for the isxo2-like sequence yielded another hit with 74 % similarity that belonged to the whole genome sequence of the P. aeruginosa ST111 outbreak strain PA38182 (Genbank - lactamase and MBL genes are annotated in this sequence; howeverHG530068.1)., no further Several information putative on β this sequence is currently available. Regarding the aac(3´)-like gene adjacent to the blaKHM-2 gene, the BLAST search yielded only a single hit and it remained unclear, why the repective ORF was annotated as an aac-type gene in the whole genome sequence of the Gloeobacter violaceus PCC 7421 strain (Genbank accession number BA000045.2). As the sequence of the putative gene showed no homologies to any other aac-like sequences in the NCBI database, it must be assumed that the annotation in the NCBI database is incorrect. Consequently, the function of the ORF downstream of the blaKHM-2 gene could not be surely identified.
A comparison of the genetic environments of blaKHM-2 and blaKHM-1 was difficult, as only little is known about the genetic environment of the KHM-1 encoding gene in the C. freundii isolate from
Japan. In the vicinity of blaKHM-1 a 360-bp ORF that encodes the hypothetical protein VP1798 of Vibrio parahaemolyticus has been described (Sekiguchi et al., 2008). This ORF was not identified in the genetic environment of blaKHM-2, so a more comprehensive comparison of the genetic contexts of both genes was not possible.
According to the literature, the blaKHM-1 gene was found only once in the C. freundii isolate from Japan (Sekiguchi et al., 2008) and no further cases where the gene was found in clinical isolate have been described since 2008. Consequently, an efficient spread of the blaKHM-1-carrying plasmid can be excluded. P. aeruginosa NRZ-03096 was referred to the NRZ from a German diagnostics lab and it is remarkable, that the only two members of a group of MBL enzymes are found in two species of a different order and with such a huge geographical distance. As KHM-2 and KHM-1 furthermore show distinct differences to each other and with respect to the large geographical distance between the two isolates, it can be speculated that KHM-2 did not originate from the Japanese KHM-1 and rather represents a de novo mobilization from an unknown environmental source of blaKHM-type genes. With a GC content of ~43 % it is furthermore implicated that KHM-type genes were mobilized into C. freundii and P. aeruginosa, which usually have higher GC contents of 51 % and 66 %, respectively. While the blaKHM-1 gene was found on a plasmid which was conjugable from C. freundii into E. coli W1895 (Sekiguchi et al., 2008), the analysis of the gene localization in this study showed that the blaKHM-2 gene was chromosomally-encoded in P. aeruginosa NRZ-03096. Consequently, the blaKHM-2 gene must have been mobilized into P. aeruginosa by a plasmid or a transposon-mediated mechanism. As two putative transposases were found adjacent to the gene, one of these could be responsible for the integration in the chromosome. Furthermore, the chromosomally localization of the gene was another indicator that the gene was mobilized into P. aeruginosa from another species, as no KHM-like genes have been described in this species so far. However, it was remarkable that the Discussion 98
isolate expressed a sequence type similar to ST395. ST395 has only rarely been described in the context of antibiotic resistance in clinical isolates. The only published cases were from France and Hungary, but ST395 was not reported as a carrier of carbapenemases in these cases (Libisch et al., 2009; Cholley et al., 2011; Slekovec et al., 2012; Valot et al., 2014). Consequently, a rapid spread of this KHM-2-carrying P. aeruginosa sequence type in healthcare settings remains questionable, as other sequence types like ST235 are significantly more prevalent worldwide.
4.4 Catalytic characteristics of IMP-31, OXA-233 and KHM-2 -lactamases are essential for their ability to confer resistance against
The catalytic-lactam properties antibiotics of β and the knowledge of kinetic properties for these enzymes is crucial forvarious correct β antibiotic therapy. The characterization of the impact of amino acid mutations on enzyme functionality is furthermore important for the understanding of the structural properties of these enzymes of high clinical importance.
4.4.1 Characteristics of IMP-31 Interestingly, E. coli TOP10 cells heterologously producing IMP-31 showed relatively low MIC increases compared to the IMP-1 strain. Although MICs for all tested carbapenems and most -lactams were distinctly increased in relation to the control strain with up to more than other128-fold β increased resistance, the total values were rather low. The IMP-1 strain showed significantly higher MICs for all tested -lactams except piperacillin, for which the MICs were only slightly increased for both IMP strainsβ . For example, the MIC for ceftazidime was 32 mg/l for the IMP-31 strain, representing one of the highest increases in relation to the control strain. For the IMP-1 strain, the MIC for ceftazidime was higher than 256 mg/l and thereby higher than detectable by Etest strips, indicating a significant difference in the catalytic properties between the two enzymes. It was shown for other IMP-type enzymes, that heterologous expression of the enzymes in E. coli led to MICs only slightly higher than for the control strains. For example, a strain expressing IMP-13 was shown to exhibit MICs of 0.125 mg/l for meropenem and ertapenem, while the IMP-1 strain showed values of 2 mg/l for both antibiotics in the respective -lactamase showed values of 0.015 mg/l, resulting in an 8- foldstudy. and The 133 control-fold increase strain without for IMP a-13 β and IMP-1, respectively (Santella et al., 2011). For a strain expressing the IMP-18 variant, MICs of 1 mg/l, 0.06 mg/l and 0.12 mg/l were detected for imipenem, meropenem and ertapenem, representing 17-fold, 4-fold and 8-fold increases in relation to the control strain. An IMP-1 expressing strain on the other hand showed a 33-fold increase in the MIC for imipenem and 133-fold MIC increases for meropenem and ertapenem in the respective study (Borgianni et al., 2011). A comparison with the MICs mediated by the next nearest relative of IMP-31, IMP-35, was not possible as no MIC data were available for this enzyme (Pournaras et al., 2013). The second nearest relative IMP-8 however was described to Discussion 99
mediate MIC increases of 15-fold for imipenem and 8-fold for meropenem, respectively (Yan et al., 2001). Compared to MIC increases mediated by the production of the third next relatives IMP-2 and IMP-19 it was noticable that expression of these enzymes resulted in significantly higher increases for imipenem than an expression of IMP-31, while the increases were similar for meropenem (Riccio et al., 2000; Neuwirth et al., 2007). With regard to the MIC increases reported from other studies, the increases mediated by production of IMP-31 remained low in comparison to the influence of an IMP-1 expression, but were nontheless higher than it was detected for several other IMP-type enzymes. The kinetic parameters that were determined for IMP-31 were in good agreement with most of the MIC data. IMP-31 showed a generally lower hydrolytic activity -lactam antibiotics than the reference enzyme IMP-1. Although IMP-31 was able to hydrolyzeagainst allβ tested penicillins, cephalosporins and carbapenems, the catalytic efficiencies were rather low, the highest value was 2.8 µM-1 s-1. In contrast, the highest hydrolysis rate for IMP-1 was 18 µM-1 s-1. The inability of IMP-31 (and∙ IMP-1) to hydrolyze the monobactam aztreonam was expected∙, as this is a key characteristic of MBL enzymes (Cornaglia et al., 2011). As no kinetic data were available for IMP-35 (Pournaras et al., 2013), a direct comparison of the kinetic parameters of IMP-31 and IMP-35 was not possible. Also no kinetic data were available for the next nearest relative IMP-8 (Yan et al., 2001). Compared to IMP-2 and IMP-19, which were the next relatives with available kinetic data, IMP-31 showed lower hydrolytic efficiencies for imipenem and meropenem than IMP-2 (Riccio et al., 2000). In comparison to IMP-19, IMP-31 showed higher rates for both antibiotics (Neuwirth et al., 2007). This was contrasting the MIC comparisons, where IMP-19 was shown to mediate a higher relative increase than IMP-31. Regarding penicillins and cephalosporins, IMP-31 showed higher catalytic efficiencies for ampicillin and ceftazidime than IMP-2. Compared to IMP-19, IMP-31 showed lower hydrolysis rates for penicllin G, but higher efficiencies against cefoxitin and ceftazidime. Although the catalytic efficiency of IMP-31 were lower than for the reference enzyme IMP-1, the high MICs of the clinical isolate P. aeruginosa NRZ-00156 indicated that even a weaker carbapenemase is apparently sufficient to confer high levels of carbapenem resistance in clinical isolates, as it has also been shown for IMP-8 , IMP-13 and IMP-18 (Yan et al., 2001; Toleman et al., 2003a; Hanson et al., 2006). Most clinical P. aeruginosa strains possess additional resistance mechanisms like porins loss, efflux pumps or -lactamases which act in concert to confer detectable carbapenem resistance additional(Livermore, β 2001). This has also been shown in this study, as the isolate P. aeruginosa NRZ-00156 carried the blaOXA-35 gene as a second -lactamase gene. It can be furthermore assumed that the expression level of genes under theβ control of a class 1 integron promoter is much higher in a wildtype strain than in an E. coli K12-derived laboratory strain such as TOP10, where both IMP-31 and IMP-1 were expressed under the control of a lac promoter, which has been described as relatively weak (Deuschle et al., 1986). Discussion 100
It has been shown for many other MBLs and IMP-type enzymes in particular, that relatively small changes in the amino acid sequence can lead to significantly altered catalytic properties. For example, IMP-10 differs by only one single amino acid substitution from IMP-1 but shows almost no hydrolysis of penicillin G and ampicillin (Iyobe et al., 2002). As IMP-31 and IMP-1 differ from each other by 50 amino acid substitutions, it was very likely that this large number of mutations had an influence on the catalytic behaviour or the tertiary structure of the enzyme. For more detailed analysis, the tertiary structure of IMP-31 was modelled using the IMP-2 crystal structure as the template, as IMP-2 shows a higher homology to IMP-31 than IMP-1. A comparison of the IMP-31 protein model and the crystal structure of IMP-1 is shown in Figure 4.2. Although the model of IMP-31 showed slight changes in the distances between the second group of zinc binding ligands and the respective zinc ion compared to IMP-1, these changes were rather low with the highest difference shown with an increase from 2.3 to 2.7 Å for His197. The distance between the two coordinated zinc ions was also only slightly changed from 3.5 to 3.6 Å but as it has been proposed that the distance between the zincs is important for the coordination of the catalytic water molecule in IMP-1 (Yamaguchi et al., 2005), this could have an influence on the hydrolytic properties. However, the quality score of the model was only 0.85 and therefore the possibility of a deviation of the model from the true structure of IMP-31 remained. As the mutations of IMP-31 are spread widely over the whole enzyme, it could also be possible that the altered characteristics result from structural changes not directly related to the active site. It is thought that substrate binding and hydrolysis of MBLs is influenced by a flexible loop near the active site which is formed by a tryptophan or phenylalanine residue (Palzkill, 2013). It has been proposed that this loop supports the tight binding of substrates and the stabilization of the
Figure 4.2 Crystal structure and homology model of the active site of IMP-1 (A) and IMP-31 (B). The highly conserved zinc binding ligands of the active site are colored in purple and the tryptophan of the flexible loop at position 81 is colored in blue. Distances between the zinc binding ligands and the two zinc ions are indicated by dashed lines and denoted in Å. The IMP-1 crystal structure was taken from PDB accession number 4UAM.2. The IMP-31 homology model was constructed using the SWISS-Model server using the crystal structure of IMP-2 (PDB accession number 4UBQ.1) as a template. The figures were rendered using PyMOL. Discussion 101
active site ligands. In IMP-type enzymes, this residue is a tryptophan located at position 46 and although this residue was not mutated in IMP-31, five surrounding residues showed alterations compared to IMP-1 (Figure 3.2). This could possibly have an influence on the function of the flexible loop and thereby influence the catalytic efficiency, as it has been shown for IMP-1 that mutations of this loop result in significantly altered turnover numbers (Moali et al., 2003). In the IMP-31 model, this loop was slightly altered and the conserved tryptophan was rotated, which could possibly affect the ability of the loop to stabilize the active site. However, further structure analysis including crystallization is needed to surely identify the reason for the catalytic behaviour of IMP-31, as these hypotheses are based on homology modelling. In conclusion, IMP-31 was shown to have a distinct carbapenemase activity that is very likely to -lactam resistance in Gram-negative clinical isolates. confer high levels of β 4.4.2 Characteristics of OXA-233 OXA-233 showed distinct diff -lactam resistance in comparison to OXA-10. While E. coli TOP10erences expressing in the ability OXA -to10 confer showed β high MICs for penicillins and penicillin/inhibitor combinations, the OXA-233 strain clearly showed an inhibition by sulbactam, tazobactam and clavulanic acid, which is very atypical for a class D enzyme, especially for OXA-10-like enzymes. OXA-type enzymes that were described as inhibited by clavulanic acid are OXA-12 (Rasmussen et al., 1994; Walsh et al., 1995), OXA-18 (Philippon et al., 1997), OXA-20 (Naas et al., 1998), OXA-45 (Toleman et al., 2003b), OXA-53 (Mulvey et al., 2004) and OXA-63 (Meziane-Cherif et al., 2008), but these do not belong to the OXA-10 group. No OXA-10-like enzymes have so far been described as inhibited by this compound. As expected, the OXA-233 expressing strain showed significantly lower MICs for piperacillin-tazobactam and amoxicillin-clavulanate than for the single antibiotics. Clavulanic acid, sulbactam and -lactam inhibitors that share structural similarity with penicillin. tazobactamWhile clavulanic are clinically acid was used isolated β from Streptomyces clavuligerus and is clinically used in the salt form clavulanate, sulbactam and tazobactam are synthetic penicillinate sulfones (Reading & Cole, 1977; English et al., 1978; Fisher et al., 1980). The mechanism of action of these substances -lactam antibiotics; however, the hydrolysis rate is extremely low, isleading similar to to an the inhibition hydrolysis of ofthe β enzyme similar to t -lactams (Drawz & Bonomo, 2010). The inhibition effectiveness is knownhe inhibition to depend of PBPs on by the β inhibitor/enzyme combination, for example TEM-1 needs 160 clavulanate molecules for inactivation, while SHV-1 requires only 60 (Drawz & Bonomo, 2010). Consequently, it can be hypothesized that the two amino acid substitions of OXA-233 relative to OXA-10 lead to a reduced amount of molecules that is necessary for inhibition. In addition, one or both of the substitutions seem to mediate the susceptibility of OXA-233 towards sulbactam, which was not detectable for OXA-10. Discussion 102
Regarding the MICs for cephalosporins, the inability of OXA-233 to confer increased resistance towards cephalotin, cefuroxime, cefoxitin, cefotaxime, ceftriaxone, cefepime and ceftazidime was remarkable, as the next nearest relative OXA-17, which differs in only one amino acid substitution to OXA-233, has been described as an extended-spectrum enzyme with high activity on cephalosporins (Danel et al., 1999). This indicated that the apparent changes in the substrate spectrum were more likely based on the V117F substitution than on N73S. Consistent with the literature, the OXA-10 strain showed only slightly increased cephalosporin resistance and no increase for ceftazidime. As expected, expression of OXA-233 led to increased MICs for carbapenems, although the increase was very low. It has been shown for several “weak” OXA -lactam hydrolysis rates, they can confer enzymes,clinically thatrelevant even resistance if they are in not Gram capable-negative of high wildtype β strains (Antunes et al., 2014). In addition, it can be assumed that expression of OXA-233 and OXA-10 in E. coli TOP10 was at a relatively low level, as the expression was not induced and controlled by a relatively weak lac promoter. Furthermore, the higher MIC increases for the OXA-233 transconjugant, where the blaOXA-233 gene was under control of the strong integron promoter, demonstrated the effect of an assumable higher expression level. As the next nearest relatives OXA-10 and OXA-17 were described as unable to hydrolyze carbapenems, it was initially assumed that the mutation at position 117 from valine to phenylalanine was responsible for the carbapenemase activity of OXA-233. But surprisingly, production of OXA-10 also led to increased MICs for carbapenems. This was remarkable, as enzymes of the OXA-10 group had always been described as narrow- or extended-spectrum enzymes with no activity on carbapenems (Poirel et al., 2010; Leonard et al., 2013; Evans & Amyes, 2014). However, in 2014 it was shown that probably all class D enzymes (including OXA-10) are in fact carbapenemases and that many previous characterizations of OXA-type enzymes were inaccurate (Antunes et al., 2014). This was explained by the fact that OXA enzymes possess a highly conserved lysine residue at position 70 (K70), which is an important part of the active site and plays a crucial role in -lactam hydrolysis. K70 is suggested to activate the attacking groups in both acylation and deacylationβ reactions during - lactam ring and therewith thought to influence -lactamasethe opening to quickly of the bind β another substrate molecule (Schneider et al., 2009the). Thisability lysine of the residue β has been shown to be carboxylated in vivo (Golemi et al., 2001) and it has further been shown that the carboxylation is required for full enzyme activity (Schneider et al., 2009). Antunes et al. (2014) stated that almost all biochemical characterizations of OXA-type enzymes were performed without the supplementation of a CO2 source to the reaction mixture and that this could lead to decarboxylation of K70, resulting in reduced enzyme activity. They further showed that the narrow-spectrum enzymes OXA-2 and OXA-10 in fact were able to hydrolyze carbapenems with low efficiency when a CO2 source was present. With this background, biochemical Discussion 103
characterization of OXA-233 and OXA-10 was performed with addition of a CO2 source to the reaction mixture in this study. Determination of kinetic parameters clearly showed that both OXA-233 and OXA-10 were able to hydrolyze carbapenems in vitro and that the carbapenemase activity was CO2-dependent, confirming the findings of Antunes and colleagues (2014). Hydrolytic efficiencies observed for OXA-233 were rather low compared to OXA-10, showing higher values for all tested substrates except for meropenem, which was the only substrate for which OXA-233 showed a higher activity. The greatest difference was detected for cephalosporins as the hydrolysis rates for cefoxitin and ceftazidime of OXA-233 were too low to be determined with the experimental setup used in this study. However, cephalosporin hydrolysis rates of OXA-10 were also extremely low. In general, the catalytic data of both OXA-233 and OXA-10 reflected the results obtained from the MIC analyses for both OXA-233 and OXA-10. Interestingly, OXA-233 showed a significantly lower affinity towards ampicillin and oxacillin and in contrast to OXA-10 was not able to hydrolyze aztreonam. This was also surprising as all other OXA-10-like enzymes have been described as beeing capable of a moderate aztreonam hydrolysis (Poirel et al., 2010). It has been mentioned before, that only two amino acid substitutions (N73S and V117F) distinguish OXA-10 and OXA-233. Consequently, at least one of these mutations must be the reason for the differences in catalytic behaviour. In all OXA enzymes except OXA-233, the highly conserved position 117 is always occupied by valine, isoleucine or rarely, leucine and is an important hydrophobic active site residue as a part of the “omega loop” (Schneider et al., 2009; Poirel et al., 2010; Leonard et al., 2013). It has been shown that mutation of V117 to aspartic acid leads to a decarboxylation of the important active site lysine K70, resulting in a loss of enzyme activity (Schneider et al., 2009). In OXA-233, position 117 is occupied by phenylalanine. The aromatic side chain of phenylalanine is more hydrophobic than the ones of valine, isoleucine or leucine, which could enhance the tertiary structure stability of the active site or the whole enzyme. It has been shown for a large variety of proteins, that increased hydrophobicity positively influences stability of the tertiary structure (Kellis et al., 1988; Pace et al., 2011). Consequently, it can be speculated that an increased hydrophobicity of the active site of OXA-233 could lead to a decreased flexibility of the site and thereby to lower hydrolysis rates. However, this would not explain several differences in the catalytic behaviours of OXA-233 and OXA-10 as the hydrolytic efficiency of OXA-233 was not overall decreased and it was slightly elevated towards meropenem and similar for ertapenem. In addition, a high hydrophobicity of the active site is beneficial for efficient carboxylation of K70 (Leonard et al., 2013) and consequently, replacement of V117 with an even more hydrophobic residue should rather result in increased enzyme activity. In site-directed mutagenesis experiments that were performed with OXA-1 by Buchman and colleagues (2012), it was shown that a substitution of V117 to
Discussion 104
Figure 4.3 Crystal structure and homology model of the active sites of OXA-10 (A) and OXA-233 (B). The highly conserved residues of the active site are colored in purple. Position 117 is colored in red. The OXA-10 structure was taken from PDB accession number 2WGW.1.B. The OXA-233 homology model was constructed using the SWISS-Model server. The figures were rendered using PyMOL. phenylalanine leads to significantly lower MICs for ampicillin when expressed in E. coli. However, lowered MICs for ampicillin were not detected for OXA-233 in this study, but as OXA-233 and OXA-1 only show a homology of 26 %, this could be based on other structural differences. To gain more detailed information on the potential structure of OXA-233 and the influence of the V117F mutation, a homology model was constructed, based on the crystal structure of OXA-10 (PDB accession number 2WGW.1.B). A comparison of the structure models of the active site of OXA-233 and OXA-10 is shown in Figure 4.3. The homology model of OXA-233 showed that the large aromatic side chain of phenylalanine extends into the space of the active site. This would definitely lead to alterations in the distances between the important active site residues (S67, K70, S115, W154, L155, K205 and G207) and F117, possibly affecting carboxylation of K70 and the catalytic behaviour. It has to be noted that modelling of the carboxylation of K70 was not possible and a comparison with the crystal structure image clearly indicates that in case of a carboxylation, the side ring of F117 and the carboxy group of K70 could collide, further implicating a possible conformational change of the active site in OXA-233. For this reason, no atom distances were calculated as they would be too inaccurate. Another explanation for the catalytic characteristics of OXA-233 could be a sterical hindrance of the
-lactams, as the measured Km values were mostly higher than detected for
OXAbinding-10. ofEspecially some β oxyimino-cephalosporin hydrolysis could be influenced, as their R2 side chains are larger than the ones of penicillins and carbapenems (Figure 4.4). This could also be an explanation for the inability of OXA-233 to hydrolyze aztreonam, which also possesses a relatively large R2 group. However, this would not explain the extremely low hydrolysis of
Discussion 105
Figure 4.4 Chemical structures of ceftazidime, aztreonam and penicillin G. Ceftazidime is shown as a representative for oxyimino-cephalosporins. The R2 side chains of oxyimino-cephalosporins and aztreonam is significantly larger than the R2 chain of penicillin G and carbapenems (Figure 1.2). cefoxitin, which possesses a R2 side chain comparable to penicillin G. To verify the hypothesis that the V177F substitution is responsible for the altered hydrolytic properties of OXA-233, further structure analysis, including crystallization and substrate binding modelling, is needed.
In conclusion, it was clearly shown that OXA-233 has a CO2-dependent carbapenemase activity -lactam antibiotics. As andOXA - that233 show this activityed extremely leads weak to increased hydrolysis resistance of cephalosporins against many, these β antibiotics could still represent a therapy option against OXA-233-producing Gram-negative pathogens. It has been proposed that oxyimino-cephalosporins are a potential therapy option for OXA-48-producing Enterobacteriaceae without an ESBL association, as like OXA-233, OXA-48-like enzymes (except OXA-163) show weak hydrolysis of these antibiotics (Poirel et al., 2012b). However, only few clinical data are available to support the use of these antibiotics with only one published study showing a successful treatment of a neonate infected by an OXA-48-producing K. pneumoniae strain in France with a combination of cefotaxim and amikacin (Levast et al., 2011). Furthermore, a sucessful treatment of a patient also infected with an OXA-48-producing K. pneumoniae strain using a combination of ceftazidime and avibactam has been reported from Spain (Mora-Rillo et al., unpublished data). Another study performed with a peritonitis model in mice also showed that treatment with ceftazidime was an efficient therapy against OXA-48- producing K. pneumoniae (Mimoz et al., 2012). Regarding these data, it can be suggested that oxyimino-cephalosporins remain an option also for OXA-233 producing Enterobacteriaceae that do not possess other cephalosporin resistance mechanisms. This is furthermore supported by the MIC data obtained for C. freundii NRZ-02127, which was shown to be susceptible or intermediate to cefotaxime, cefepime and ceftazidime, indicating potential therapy options against producers of this novel class D carbapenemase. In addition, the observed inhibition by clavulanic acid in the MIC studies might be clinically relevant, but has to be further analyzed in future experiments. Discussion 106
4.4.3 Characteristics of KHM-2 An expression of KHM-2 in E. coli - lactams except piperacillin and aztreonam.TOP10 resulted Compared in significantly to the only higherknown MICs relative for , allKHM tested-1, the β resistance spectrum showed higher MIC increases for ampicillin and amoxicillin, indicating a more efficient hydrolysis of these substrates. Cephalosporin MICs were on a comparable level for both strains. Regarding carbapenems, MICs were also elevated for both strains, with the KHM-2 strain showing a 2-fold higher increase for imipenem, while the KHM-1 strain showed higher increases for meropenem, doripenem and ertapenem. The MICs which were detected for both strains were significantly higher than compared to those of the control strain with increases of up to 8,000-fold for cefotaxime. The kinetic analysis of KHM-2 and KHM-1 reflected the resistance spectrum observed in the MIC studies for most tested substrates with penicillin G, cefoxitin and ceftazidime beeing well hydrolyzed with efficiencies of up to 10.5 µM-1 s-1.
Interestingly, the hydrolysis rate of KHM-2 for imipenem was higher than the rate ∙ for ceftazidime, while it was distinctly lower for the reference enzyme KHM-1. Consistent with the MIC data, KHM-1 had higher rates for meropenem and ertapenem. The greatest difference in catalytic efficiency was detected for cefotaxime, which was also 10-fold more efficiently hydrolyzed by KHM-1. Like all other MBLs characterized in this study, KHM-2 was not able to hydrolyze aztreonam. As no information is available in the literature regarding the structure or specific amino acids of KHM-1 and as this enzyme furthermore shows a homology of only 74 % to KHM-2, it was difficult to form a hypothesis for the influence of the mutations of KHM-2 -lactam hydrolysis.
As the zinc binding ligands of KHM-2 and KHM-1 are identical to other subclasson β B1 MBls (Garau et al., 2004) and were not altered in KHM-2, a weaker zinc binding, possibly influencing the hydrolytic efficiency, is rather unlikely. However, the substitution at position 100 from threonine to aspartic acid could have an influence on the zinc coordination. The first zinc binding site of subclass B1 MBLs is formed by two histidines at the consensus positions 116, 118 and 196, while the second zinc binding site is formed by an aspartic acid residue at postion 120, a cysteine at position 221 and a histidine at position 263 according to the MBL standard numbering scheme (Garau et al., 2004). In the KHM-1 and KHM-2 sequences, these residues correspond to the positions 97, 99 and 159 for the first binding site and 101, 178 and 217 for the second binding site. For KHM-2, the T100D subsitution leads to two neighbored aspartic acid residues, which might compete as zinc-binding ligands. This could result in an alteration of the distances between the two zinc ions or the three ligands of the second zinc binding site. As these residues play a crucial role for correct coordination of the zinc ions for the nucleophilic attack on -lactam ring (Palzkill, 2013), a distance alteration could possibly affect the hydrolytic theefficiency β . To gather more information on the putative influences of the mutations and to substantiate these hypotheses, the structures of KHM-2 and KHM-1 were modelled based on Discussion 107
homologies with the crystal structure of IMP-1, which was the next nearest relative to KHM-type MBLs, but with an identity of only 58.33 % to KHM-1 and 61.36 % to KHM-2. The homology models are shown in Figure 4.5. Modelling showed that the zinc binding site of KHM-2 could in fact be influenced by the T100D substitution, as the zinc binding sites of both aspartic acid residues are oriented in the same direction, which could lead to disturbances in zinc coordination. This could lead to altered hydrolytic characteristics, as it has been shown for IMP-1 that mutations of Asp120 (MBL standard numbering scheme; Asp101 in KHM-type enzymes) can significantly influence the hydrolytic efficiency due to alterations in the distance between the two coordinated zinc ions (Yamaguchi et al., 2005). Regarding the conserved tryptophan of the flexible loop, the models showed that the mutations of the surrounding residues in KHM-2 probably lead to a conformational change of the loop, resulting in an increased distance of the tryptophan to the active site. As it has been shown for IMP-1, mutations of this residue can affect the kcat values for various substrates (Moali et al., 2003). Consequently, the increased distance to the active site in KHM-2 could influence the hydrolytic characteristics of KHM-2. However, as the model qualities were rather low due to the low homology to the template crystal structure, these conclusions remain hypothetical and have to be confirmed by crystal structure analysis of both KHM-2 and KHM-1. As the differences in substrate hydrolysis between KHM-2 and KHM-1 were furthermore rather diverse with some rates beeing higher and some lower for KHM-2, these can not be fully explained on the basis of
Figure 4.5 Homology models of KHM-1 (A) and KHM-2 (B). The highly conserved zinc binding ligands of the active site are colored in purple for the first ligand group and in blue for the second ligand group. The T100D substitution in KHM-2 is colored in cyan and the conserved tryptophans of the flexible loop of MBLs are colored in orange. Both models were constructed using the SWISS-Model server. The crystal structure of IMP-1 (PDB accession number 1ddk.1) which was the next nearest related structure (58.33 % homology to KHM-1 and 61.36 % to KHM-2) was used as a template. The figures were rendered using PyMOL. Discussion 108
the models as the mutations mentioned here mostly led to overall lowered or increased activity in IMP-1 (Moali et al., 2003; Yamaguchi et al., 2005). In conclusion, it was shown that KHM-2 is a novel metallo- -lactamase with a high carbapenemase activity, showing distinct differences to the next nearestβ relative KHM-1 in catalytic behaviour. It can be hypothesized that these altered characteristics are based on several mutations of amino acid residues in the vicinity of the highly conserved residues of the zinc binding motifs and the flexible loop of subclass B1 MBLs.
4.5 Characterization of the blaOXA-233-carrying plasmid pMB3018 The plasmid pMB3018 was related to the four other IncN-like plasmids pJIE137, p271A, pECS01 and pTR3. The plasmid backbone of all five plasmids showed high homologies and it has been suggested that this variant of the IncN-type be classified as a novel subgroup named IncN2 (Poirel et al., 2011). The main characteristic of the other IncN2-type plasmids is the presence of a complete tra locus with the tra genes K, J and I beeing separated from the main locus. These genes code for subunits of the sex pilus or for proteins with various functions necessary for conjugational transfer, e.g. plasmid stability proteins or components of the relaxosome (Zatyka & Thomas, 1998). In pJIE137 the traKJI-locus is located downstream of the main tra-locus, separated by the ΔfipA gene (Partridge et al., 2012), which was also found in pMB3018. In p271A and pTR3 the traKJI-locus is located more distant to the main locus on the 3´-extremity of the oriT (Poirel et al., 2011; Chen et al., 2012), showing one of the main differences regarding the plasmid backbone to pMB3018. A region that was found in all five related plasmids was the region containing the stbA, stbB and stbC genes next to the traKJI-locus, which are predicted to code for plasmid stability proteins. While p271A, pTR3 and pECS01 were described as carrying the blaNDM-1 gene, bracketed by the two insertion sequences ISEc33 and ISSen4, pJIE137 carries a blaCTX-M-16 gene and is missing the insertion sequences (Partridge et al., 2012). In pMB3018 however, ISSen4 was present, but not associated with any resistance gene and without its counterpart ISEc33. It can be hypothesized that the blaNDM-1-carrying insertion sequence was disrupted by other mobile genetic elements in pMB3018, as several ORFs coding for phage integrases or hypothetical transposases were located next to ISSen4. This was supported by the absence of repeat sequences that could be correlated to ISSen4, as the second repeat could have been deleted during an integration of the phage integrase genes. Another option could be an incomplete excision of the NDM-1 IS, resulting in the remaining ISSen4. pJIE137 possesses a 5.2- kb region that corresponds to the CUP (conserved upstream repeat)-controlled regulon of the IncN plasmid R46 (Delver & Belogurov, 1997). This region consists of the ardR, ardB and ardK genes, coding for antirestriction proteins, a gene coding for a single-strand DNA binding protein (ssb) and the repA gene. While Ard proteins provide protection from the restriction enzymes of the recipient during conjugation (Wilkins, 2002), the single-strand binding protein is predicted Discussion 109
to have a protective function in conjugation which is not known in detail (Delver & Belogurov, 1997). In contrast to pJIE137, the plasmids p271A, pTR3 and pECS01 contain a partially deleted CUP region, missing the ardR and ardB genes. In pMB3018, all genes of the CUP region of pJIE137 were identified but were disrupted by the large putative transposon or IS structure that contained the two putative phage integrase genes and ISSen4. As this region showed a significantly lower GC content compaired to the rest of the plasmid, it can be assumed that it represents the result of an integration event of mobilized DNA from an unknown source. This contradicts the hypothesis of the disrupted NDM-1 IS and rather indicates that the ISSen4 in pMB3018 represents an independent insertion event, as the NDM-1 IS in p271A, pTR3 and pECS01 is not neighbored to the CUP region (Poirel et al., 2011; Chen et al., 2012; Netikul et al.,
2014). A unique feature of pMB3018 was the blaOXA-233-carrying class 1 integron which was neither found in pJIE137 nor in the other three related plasmids. Although pJIE137 carried a - classlactamase 1 integron, genes it( Partridgewas located et atal. a, 2012different). The region blaOXA than-233 -incarrying pMB3018 integron and did was not neighboredcontain any byβ IS6100, but as IS6100 was bracketed by two 123-bp inverted repeats it possibly represented a complete insertion sequence element. However, the identification of a 13-bp inverted repeat bracketing the integron and IS6100 was consistent with a perfect transposon structure and it is as well possible that the blaOXA-233 integron was mobilized into pMB3018 by this structure. In conclusion and regarding the differences to pJIE137 and the p271A-like plasmids, it is more likely that pMB3018 originated from pJIE137 or a common ancestor, as the two plasmids shared the same organization of the two tra loci and the CUP region, which however was disrupted by a large putative transposon in pMB3018. The blaOXA-233 gene was a unique feature of pMB3018 and no related integron structure was found in any other IncN2 plasmid deposited in the NCBI -lactamase gene carrying genetic structures. database, demonstrating the immense diversity of β 4.6 Comparison of IMP-31, KHM-2 and OXA-233 and concluding remarks Compared to each other, the three novel carbapenemase described in this study showed significantly different substrate profiles. The metallo- -lactamases IMP-31 and KHM-2 showed an efficient hydrolysis of penicillins, cephalosporins andβ carbapenems as it has been described as a common characteristic of MBL enzymes and represents the most clinically relevant feature -lactamases (Walsh et al., 2005; Gupta, 2008b; Cornaglia et al., 2011). In most cases,of this thegroup presence of β of an MBL in a clinical Gram-negative pathogen is equivalent to the almost -lactam antibiotics for therapy. As MBL genes are very often completeaccompanied exclusion by additional of β resistance mechanisms against various classes of antibiotics, MBL- producing isolates can easily become pan-resistant (Maltezou, 2009). This is further aggravated by the fact that no MBL inhibitors are available for clinical use (Drawz & Bonomo, 2010). Although it has been shown for many metallo- -lactamase that these enzymes are not able to
β Discussion 110
hydrolyze piperacillin with high efficiencies, this was never stated as a common characteristic of MBLs (Walsh et al., 2005; Gupta, 2008b; Maltezou, 2009; Cornaglia et al., 2011). As all four MBLs analyzed in this study showed very low hydrolysis rates for penicillin and as the KHM-2- harbouring isolate P. aeruginosa NRZ-03096 was susceptible to piperacillin according to EUCAST criteria, this antibiotic might still represent a possible treatment option, even in the presence of an MBL. However, this could only be an option in the case of the absence of additional piperacillin resistance mechanisms such as porin loss, exporter pumps or expression of an -lactamase. -lactams, both IMP-31 and KHM-2 showed a distincothert effect β on the resistanceRegarding of E.all coli other, but tested it was β remarkable that the KHM-producing strains showed significantly higher MIC increases than the IMP and OXA expressing strains. This either -lactam antibiotics by KHM-2 and KHM-1 or a indicatedsignificantly a higher very efficient expression hydrolysis in E. coli TOP10 of β than the IMP- and OXA-expressing strains. But as the kinetic parameters of both KHM-type enzymes were not very different from the IMP and OXA-type enzymes characterized in this study, this indicated a significantly increased expression in E. coli TOP10. However, the specific reason for this assumed higher expression remains unclear. In conclusion, both IMP-31 and KHM- -lactam resistance and it must be assumed that both enzymes are 2able had to a confersignificant clinically impact relevant on β resistance levels -lactams and carbapenems in particular in Gram-negative species of clinical importance. In forcontrast β to the broad spectrum of IMP-31 and KHM-2, the class D enzyme OXA-233 showed very low hydrolysis of cephalosporins but was able to hydrolyze carbapenems if supplied with a CO2 source. As sufficient CO2 sources are probably abundant at infection sites, the experimental conditions used in this study likely reflect the situation in vivo and it can be assumed that the confirmation of a carbapenemase activity of OXA-10-like enzymes might be clinically relevant. In conclusion, it was shown for all three discovered -lactamases that they possess a
novel-lactam β resistance in E. coli, including distinctresistance carbapenemase to carbapenems. activity As theand OXAcan confer-233-encoding increased gene β was located on a transconjugable plasmid and as the blaIMP-31 and blaKHM-2 genes were very likely located in transposon structures on the chromosome, it can be furthermore assumed that all three enzymes are able to spread into other organisms and might play an important role in the future for multidrug-resistance in Gram-negative pathogens.
Summary 111
5 Summary
The increasing number of carbapenemase-producing Gram-negative pathogens responsible for healthcare-associated infections is a major clinical problem. Consequently, the identification and characterization of novel carbapenemase genes and their encoded enzymes is crucial for both clinical diagnostics and antimicrobial therapy. In this thesis, three carbapenem-resistant Gram- negative clinical isolates of the species Pseudomonas aeruginosa and Citrobacter freundii were analyzed on the presence of a novel carbapenemase and three novel enzymes were successfully discovered by PCR techniques and shotgun cloning approaches: IMP-31, OXA-233 and KHM-2. While IMP-31 and KHM-2 were metallo- - -lactamase class B,
OXA-233 was an OXA-10 related class D enzyme.β lactamases By characterization of the molecular of the βgenetic environment -lactamase genes and analysis of the gene localization it was shown that ofbla theIMP- 31three and novelblaOXA -β233 were part of a class 1 integron, while blaKHM-2 was not part of such a genetic structure. With pulsed-field gel electrophoresis experiments and Southern blot hybridizations, it was shown that the IMP-31 and KHM-2 encoding genes were located on the bacterial chromosome of the clinical isolates, implying that the two MBL genes were integrated into the respective chromosome by a transposon-mediated mechanism. On the other hand, the blaOXA-233 gene was identified on the conjugable plasmid pMB3018 with a size of 52 kb which was fully sequenced by 454-pyrosequencing and was shown to be a member of the IncN2 incompatibility group. As the genes identified in this study showed a significantly different GC content compared to the species they were found in, it must be assumed that they were mobilized into these species from a still unknown source. The impact of a production of IMP-31, OXA-233 and KHM-2 -lactam resistance was analyzed by determination of the minimal inhibitory concentrationon β(MIC) for -lactam antibiotics for Escherichia coli strains expressing the three novel enzymesvarious and their β respective reference enzymes. The analysis showed that production of all three enzymes leads to significantly increased resistance against most -lactams and carbapenems in particular. By determination of the kinetic parameters kcat and Kβm, which reflect the catalytic efficiency of an enzyme, FPLC-purified IMP-31, OXA-233, KHM-2 and their respective reference enzymes were characterized biochemically by in vitro hydrolysis assays - lactam substrates. Forusing each non enzyme,-linear regression the kinetic andparameters the analyses were showeddetermined that forall tenthree different enzymes β are distinct carbapenemases which are likely to confer clinically relevant carbapenem resistance levels in Gram-negative pathogens. IMP-31 and KHM-2 showed hydrolysis of almost every tested -lactam, while OXA-233 was lacking a high hydrolytic efficiency for cephalosporins, indicating a
βpossible treatment option. Carbapenem hydrolysis of OXA-233 was CO2 dependent and confirmed the recent finding, that probably all class D enzymes are able to hydrolyze Summary 112
carbapenems when supplied with a CO2 source. The identification and characterization of IMP-31, OXA-233 and KHM-2 in this thesis underlines the ongoing spread and diversification of carbapenemases in Gram-negative species of clinical importance. Zusammenfassung 113
6 Zusammenfassung
Die zunehmende Zahl von Carbapenemase-produzierenden Gram-negativen Krankheitserregern ist ein immenses klinisches Problem. Daher ist die Identifizierung und Charakterisierung neuer Varianten dieser Enzyme von großer Bedeutung für die klinische Diagnostik und die korrekte Antibiotikatherapie. In dieser Arbeit wurden drei klinische Isolate der Spezies Pseudomonas aeruginosa und Citrobacter freundii mittels diverser PCR-Techniken und Shotgun- Klonierungsexperimenten auf das Vorhandensein von neuen Carbapenemasen hin untersucht. Hierbei konnten drei neue Enzyme identifiziert werden: IMP-31, OXA-233 und KHM-2. Während IMP-31 und KHM-2 Metallo- -Laktamasen der molekularen Klasse B waren, stellte OXA-233 ein
OXA-10-ähn β -Laktamasen dar. Durch die Charakterisierung der liches Mitglied der Klasse D - βLaktamasegene konnte gezeigt werden, dass sowohl dasgenetischen blaIMP-31- Umgebungals auch das der bla dreiKHM- 2 neuen-Gen Bestandt β eil eines Klasse 1 Integrons waren, während das blaOXA-233-Gen nicht in einer derartigen genetischen Struktur vorlag. Mittels Pulsfeldgelektrophoreseanalysen, gefolgt von Southern Blot-Hybridisierungen, konnte gezeigt werden, dass die IMP-31- und KHM-2-kodierenden Gene waren auf dem Chromosom des jeweiligen Isolats lokalisiert waren, was auf eine Transposon-vermittelte Integration schließen ließ. Das OXA-233-kodierende Gen hingegen wurde auf dem konjugierbaren Plasmid pMB3018 lokalisiert, welches eine Größe von 52 kb aufwies. Dieses Plasmid wurde im Rahmen dieser Arbeit mittels 454-Pyrosequencing komplett sequenziert und die Sequenzanalyse ergab, dass dieses Plasmid zum IncN2-Inkompatibilitätstyps gehört. Da die in dieser Studie identifizierten Gene in ihrem GC-Gehalt deutlich von dem der Spezies, in denen sie identifiziert wurden, abwichen, muss davon ausgegangen werden, dass diese Gene von einer bislang unbekannten Quelle in diese Spezies mobilisiert wurden. Der Einfluss von IMP-31, OXA-233 und KHM-2 auf -Laktamantibiotika wurde mittels Bestimmung der minimalen
Hemmkonzentrationdie Resistenz gegenüber (MHK) β für Escherichia coli-Stämme, die die entsprechenden Enzyme exprimierten, untersucht. Die Analyse ergab, dass alle drei Enzyme eine deutlich erhöhte -Laktame und damit auch gegen Carbapeneme vermittelten. Durch die
ResistenzBestimmung gegen der kinetischen β Parameter kcat und Km, welche ein Maß für die katalytische Effizienz eines Enzyms darstellen, wurden die drei neuen Carbapenemasen und ihre jeweiligen Referenzenzyme mittels FPLC aufgereinigt und in in vitro Hydrolyseuntersuchungen biochemisch charakterisiert. Die Bestimmung der kinetischen Parameter zeigte, dass die drei Enzyme eine deutliche Carbapenemaseaktivität aufwiesen und es damit höchst wahrscheinlich ist, dass diese Enzyme eine hohe und damit klinisch relevante Carbapenemresistenz in Gram- negativen Erregern vermitteln können. Für jedes der sechs Enzyme wurden die kinetischen Parameter für zehn verschiedene Substrate mittels nichtlinearer Regression bestimmt und es Zusammenfassung 114
zeigte sich, dass IMP-31 und KHM-2 in der Lage waren, nahezu alle Substrate mit hoher Effizienz zu hydrolysieren, während OXA-233 keine hohe hydrolytische Aktivität gegenüber Cephalosporinen besaß. Dies könnte auf eine mögliche Therapieoption hindeuten. Des Weiteren war die Carbapenemhydrolyse von OXA-233 CO2-abhängig, was die erst vor Kurzem formulierte
Annahme -Laktamasen eine CO2-abhängige
Carbapenemasefunktion, dass möglicherweise besitzen, untermauerte. alle Klasse Die DIdentifizierung β und Charakterisierung von IMP-31, OXA-233 und KHM-2 im Rahmen dieser Arbeit ist ein weiteres Indiz für die fortwährende Verbreitung und Diversifikation von Carbapenemasen in Gram-negativen Spezies mit klinischer Relevanz.
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Appendix 132
8 Appendix
Appendix 1 Ion exchange (A) and gel filtration (B) chromatograms of the IMP-31 FPLC. Shown are the chromogram curves monitored during the chromatography process. The curves show the amount of detected milli absorbance units (mAU) relative to the volume passed through the columns. Red lines indicate the collected fractions. The ion exchange step for IMP-31 was performed at a pH of 7.5.
Appendix 133
Appendix 2 Ion exchange (A) and gel filtration (B) chromatograms of the IMP-1 FPLC. Shown are the chromogram curves monitored during the chromatography process. The curves show the amount of detected milli absorbance units (mAU) relative to the volume passed through the columns. Red lines indicate the collected fractions. The ion exchange step for IMP-1 was performed at a pH of 7.5.
Appendix 134
Appendix 3 Ion exchange (A) and gel filtration (B) chromatograms of the OXA-233 FPLC. Shown are the chromogram curves monitored during the chromatography process. The curves show the amount of detected milli absorbance units (mAU) relative to the volume passed through the columns. Red lines indicate the collected fractions. The ion exchange step for OXA-233 was performed at a pH of 6.0.
Appendix 135
Appendix 4 Ion exchange (A) and gel filtration (B) chromatograms of the OXA-10 FPLC. Shown are the chromogram curves monitored during the chromatography process. The curves show the amount of detected milli absorbance units (mAU) relative to the volume passed through the columns. Red lines indicate the collected fractions. The ion exchange step for OXA-10 was performed at a pH of 4.9.
Appendix 136
Appendix 5 Ion exchange (A) and gel filtration (B) chromatograms of the KHM-1 FPLC. Shown are the chromogram curves monitored during the chromatography process. The curves show the amount of detected milli absorbance units (mAU) relative to the volume passed through the columns. Red lines indicate the collected fractions. The ion exchange step for KHM-1 was performed at a pH of 7.5.
Publications 137
Publications
Research articles
Pfennigwerth N, Geis G, Gatermann SG, Kaase M (2015) Description of IMP-31, a novel metallo- -lactamase found in an ST235 Pseudomonas aeruginosa strain in Western Germany.
J Antimicrobβ Chemother. [Published online ahead print, pii: dkv079]
Conference posters
Pfennigwerth N, Gatermann SG, Korte M, Neumann S, Marlinghaus L, Kaase M (2011) Description of a novel IMP carbapenemase, IMP-31, in two Pseudomonas aeruginosa isolates from Germany. Poster ERP08. 63. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM), Essen, Germany
Pfennigwerth N, Gatermann SG, Kaase M (2012) Description of IMP-31, a novel metallo- - lactamase very divergent from other known IMP carbapenemases. Poster P1237. β 22nd European Congress for Clinical Microbiology and Infections Diseases (ECCMID), London, UK
Pfennigwerth N, Meining L, Lang R, Gatermann SG, Kaase M (2012) Description of OXA-233, -lactamase with activity against carbapenems. Poster PRP06. a64. novel Jahrestagung class D β der Deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM), Hamburg, Germany
Pfennigwerth N, Meining L, Lang R, Gatermann SG, Kaase M (2013) Description of OXA-233, a novel class D carbapenemase inhibited by clavulanic acid. Poster P1278. 23th European Congress for Clinical Microbiology and Infections Diseases (ECCMID), Berlin, Germany
Pfennigwerth N, Gatermann SG, Kaase M (2013) Phylogenetic analysis of the IMP-type carbapenemase IMP-31 from Pseudomonas aeruginosa. Poster PRP05. 65. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM), Rostock, Germany Publications 138
Pfennigwerth N, Hoffmann A, Belmar-Campos C, Gatermann SG, Kaase M (2014) Description of KHM-2, a novel metallo- -lactamase found in a clinical Pseudomonas aeruginosa isolate from Germany. Poster P1120. β 24th European Congress for Clinical Microbiology and Infections Diseases (ECCMID), Barcelona, Spain
Pfennigwerth N, Hoffmann A, Belmar-Campos C, Gatermann SG, Kaase M (2014) Description of KHM-2, a novel metallo- -lactamase found in a clinical Pseudomonas aeruginosa isolate from Germany. Poster PRP42. β 66. Jahrestagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM), Dresden, Germany
Curriculum vitae 139
Curriculum vitae
Name Niels Ernst Pfennigwerth Date of birth 26.05.1985 Place of birth Essen Nationality German Family status married
Education
since 04/2011 PhD studies at the Department of Medical Microbiology Ruhr-University Bochum, Prof. Dr. Sören G. Gatermann Title: Identification and characterization of novel carbapenemases
10/2008-04/2011 Master studies in Biology Ruhr-University Bochum
Master thesis Department of Microbial Biology Ruhr-University Bochum, Prof. Dr. Franz Narberhaus Title: Analysis of the stability and localisation of the KDO transferase KdtA from Escherichia coli
10/2005-09/2008 Bachelor studies in Biology Ruhr-University Bochum
Bachelor thesis Department of General and Molecular Botany Ruhr-University Bochum, Prof. Dr. Ulrich Kück Title: Synthesis of Raa4 from Chlamydomonas reinhardtii in E. coli for in vitro binding studies
08/1991-05/2004 Gymnasium Essen-Überruhr Graduation: Diploma from German secondary school qualifying for university admission or matriculation
Other activities
01/2008-02/2008 Internship at the microbiological lab ZLM GmbH, Essen Erklärung 140
Erklärung
Hiermit erkläre ich, dass ich die Arbeit selbstständig verfasst und bei keiner anderen Fakultät eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel verwendet habe. Es handelt sich bei der heute von mir eingereichten Dissertation um sechs in Wort und Bild völlig übereinstimmende Exemplare. Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und in keinem Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.
Bochum, den
______Niels Ernst Pfennigwerth