In vitro dynamics and mechanisms of resistance
development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa
Maria Antonia Gomis Font
Master’s Thesis
Master’s degree in Advanced Microbiology
(With a speciality/Itinerary Research in Clinical Microbiology) at the
UNIVERSITAT DE LES ILLES BALEARS
Academic year 2018-2019
Date September 17th, 2019
UIB Master’s Thesis Supervisor Antonio Oliver Palomo
INDEX
1. Abstract……….……….…………5
2. Introduction a. P. aeruginosa lineage………7
b. P. aeruginosa characterization ……….………7
c. P. aeruginosa in patients with respiratory infections………8
d. P. aeruginosa resistance mechanisms i. Intrinsic resistance mechanisms……….…...9
ii. Acquired resistance mechanisms……….11
e. P. aeruginosa in vitro treatment……….………….12
f. Imipenem/relebactam mechanism of action………13
g. Imipenem/relebactam treatment i. Pharmacokinetics and pharmacodynamics………..13
ii. Animal studies……….………14
iii. Clinical trials……….………...14
iv. Place of imipenem/relebactam in therapy………...…....…15
3. Hypothesis……….……….………...15
4. Objectives……….……….……….….15
5. Materials and methods a. Strains i. PAO1……….………..16
ii. PAOMS……….………..16
b. In vitro resistance development……….……..……16
c. Susceptibility testing i. Sensititre ® custom plates………...18
ii. MHB microdilution……….………19
d. Characterization of resistance mechanisms
i. Library preparation and whole genome sequencing……….19
ii. Variant calling……….……….20
e. C. elegans killing assay………21
6. Results a. Dynamics of resistance development to imipenem and imipenem/relebactam……...…...…...22
b. Inverse correlation between resistance and virulence in the C. elegans model……….………..23
c. Mutants characterization by WGS……….………..24
7. Discussion.……….………..27
8. Conclusions……….……….………...28
9. Bibliography……….……….………..28
ABSTRACT
Pseudomonas aeruginosa, is a major cause of nosocomial and chronic infections, being considered a paradigm of antimicrobial resistance development. However, in the present study the dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in wild-type (PAO1) and mutator P. aeruginosa (PAOMS, ΔmutS) have been compared. The strains were incubated in Müeller-Hinton Broth (MHB) for 24h with 0.125 to 64 µg/mL of imipenem, and imipenem/relebactam at 4 µg/mL fixed concentration. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 µg/mL of imipenem for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed for 40 derived mutants from PAO1 and 40 from PAOMS. Those mutants were further characterized by whole-genome sequencing (WGS) through DNA isolation, and its virulence studied by Caenorhabditis elegans model. Development of high-level imipenem resistance development was faster for PAOMS than PAO1 mutants.
Furthermore, when both strains were treated with imipenem/relebactam, development resistance to imipenem was reduced. Moreover, characterization of mutants by WGS indicated that most of mutants which were resistant to imipenem, had mutations on oprD and ampC, while imipenem/relebactam resistant mutants had mutations in oprD and MexAB-OprM efflux system genes. Additionally, virulence and lethality of PAO1 and PAOMS mutants tested by C. elegans model demonstrated that those more resistant mutants were non-virulent, and the more sensitive ones were considered virulent. Those results suggested relebactam as an adequate partner for imipenem to remedy infections caused by P. aeruginosa resistant to carbapenems.
Key words: P. aeruginosa, resistance development, imipenem, relebactam, virulence, C.
elegans.
P. aeruginosa es una de las mayores causas de infecciones nosocomiales y crónicas, siendo un paradigma del desarrollo de resistencias a antibióticos. Así pues, en el presente estudio se compara la dinámica y el desarrollo de mecanismos de resistencia a imipenem e imipenem/relebactam en cepas de P. aeruginosa PAO1, así como de P.
aeruginosaΔmutS conocida como PAOMS. Ambas cepas fueron incubadas en caldo Müeller-Hinton por separado durante 24h, a una concentración creciente de imipenem de
0.125 a 64 µg/mL, e imipenem/relebactam a concentración constante de este último de 4 µg/mL. Los tubos de mayor concentración de antibiótico con crecimiento visible fueron reinoculados en medio fresco con concentraciones superiores de imipenem, repitiéndose el mismo procedimiento durante 7 días consecutivos. Los perfiles de sensibilidad y mecanismos de resistencia se estudiaron de cada uno de los mutantes obtenidos, siendo caracterizados por secuenciación del genoma entero a partir del aislamiento de ADN.
Además, su virulencia fue estudiada mediante la infección del modelo C. elegans. En los resultados se observó que los mutantes derivados de PAOMS desarrollaron resistencia a imipenem antes que los derivados de PAO1 cuando habían sido sometidos a presión selectiva con este mismo antibiótico. Asimismo, cuando ambas cepas fueron tratadas con imipenem/relebactam, el desarrollo de resistencia frente a imipenem fue reducido. La caracterización de los mutantes mediante la secuenciación del genoma entero resaltó que los mutantes resistentes a imipenem poseían mutaciones en los genes relacionados con la hiperproducción de la cefalosporinasa AmpC y la porina OprD, en cambio, aquellos mutantes resistentes a imipenem/relebactam demostraron mutaciones en el sistema de eflujo MexAB-OprM así como también en oprD. Finalmente, el estudio de la virulencia en C. elegans demostró que aquellos mutantes más resistentes fueron menos virulentos, e inversamente los mutantes menos resistentes resultaron virulentos. Estos resultados sugieren que el relebactam es la combinación más adecuada junto con el imipenem para remediar infecciones causadas por P. aeruginosa resistente a carbapenémicos.
Palabras clave: P. aeruginosa, desarrollo de resistencia, imipenem, relebactam, virulencia, C. elegans.
INTRODUCTION P. aeruginosa lineage
P. aeruginosa is a cellular organism who belongs to the Bacteria domain, Proteobacteria phylum, Gammaproteobacteria class, Pseudomonadales order, Pseudomonaceae family, Pseudomonas genus and P. aeruginosa group (Garrity et al., 2007).
P. aeruginosa characterization
P. aeruginosa is the most pathogenic species in the Pseudomonadaceae family. It is a non-sporulate, Gram-negative, straight or curved rod with a length from 1 to 3 µm. This pathogen produces cell surface fimbriae or pili and polar flagellum, which confer its motility. Most isolates are recognizable due to its colonial morphology, smell and production of hydrosoluble pigments (pyocyanin, pyorubin, pyomelanin and/or pyoverdine). Colonies are usually flat, spreading and have a serrated border, but also other morphologies exist. This pathogen is able to metabolize a vast diversity of carbon sources, but it does not ferment carbohydrates. It produces acid from sugars such as glucose, fructose and xylose, but not from lactose or sucrose. Also, it is positive in oxidase, catalase and arginine tests. Moreover, it grows best aerobically but can also grow anaerobically. Although its optimal temperature is 37ºC, but it can also grow at 42ºC. To sum up a brief characterization, it is important to highlight that P. aeruginosa has a non- clonal epidemic population structure, where a limited number of disseminated clones have been selected from a high number of clones (Oliver et al., 2015).
P. aeruginosa is able to colonize a wide diversity of ecological niches such as water, soil, animals, plants, and humans, where it is able to infect or be part of normal microbiota.
This bacterium is one of the most relevant pathogens causing human opportunistic infections due to its metabolic and genomic plasticity and versatility (Gellatly and Hancock, 2013). Furthermore, it is essential to underline that P. aeruginosa is one of the most recurrent and severe causes of acute nosocomial infections in patients with respiratory diseases or severe wounds (Vincent, 2003).
The flexibility of this bacteria is conferred by its large proportion of regulatory, virulence and resistance genes which are encoded in its wide genome (>6 Mb), converting P.
aeruginosa as one of the most worldwide feared pathogens (Gellatly and Hancock, 2013).
Thus, P. aeruginosa is intrinsically resistant to almost all antipseudomonal antibiotics due to an inherent antibiotic resistance machinery, based on three fundamental aspects: ampC hyperexpression, constitutive or inducible expression of efflux pumps and reduced permeability of its outer membrane via OprD porin inactivation (Lister et al., 2009). Also, antibiotic resistance in P. aeruginosa can be effectively acquired either by mutations in intrinsic chromosomal genes, or also by the horizontal transfer of resistance determinants (Livermore, 2002). Both mechanisms acquired and inherent are frequently found in hospital isolates, which resistance rates are extremely high due to the extraordinary capacity of this pathogen to accumulate mutations. Furthermore, mutation-mediated antibiotic resistance development during antibiotic treatment contributes to therapy failure as it has been mentioned during the last decades by several researchers (Fish et al., 1995; Wargo et al., 2015).
P. aeruginosa in patients with respiratory infections
Chronic and acute respiratory infections caused by P. aeruginosa have their major problematics in cystic fibrosis (CF) patients, due to the presence of hypermutable strains.
Recent studies have shown statistical link between hypermutation and antibiotic resistance, and also, genetic adaptation these extreme environments, such as CF patients’
lungs (Oliver, 2010).
P. aeruginosa hypermutable strains are those which its mismatch repair (MMR) system is truncated. Mismatches during DNA replication or homologous recombination are repaired by different mechanisms. MMR is started by the MutS, MutL and MutH proteins.
At the beginning, MutS recognizes a mismatched base pair as well as insertions or deletions (1→4 nucleotides). Then, MutL forms a complex with MutS activating the MutH endonuclease. After, the DNA helicase II ensures the separation of DNA strands.
So, the inactivation of the MMR system leads to an increase in the mutation rate due to the inability to repair mismatches correctly. The inactivation of any involved MMR genes (mutS, mutL, mutH and uvrD or mutU) increases the rate of mutation from 100 to 1000- fold (Modrich and Lahue, 1996). It is important to highlight the presence of high
proportion of P. aeruginosa isolates with an extremely high mutation frequency (mutators) in CF patients’ lungs (20%) due to its ability to adapt to extreme environments (Oliver et al., 2002).
Hypermutation has seemed to be a P. aeruginosa common feature in chronically infected CF patients and it has been found to be linked with antibiotic resistance development (Oliver et al., 2000). Furthermore, hypermutation appears not to be frequent in P.
aeruginosa isolates from acute infections (Oliver et al., 2000). The problematics of these acute infections is the horizontal gene transfer between P. aeruginosa isolates in hospitals (Montero et al., 2019) where antibiotic treatments act as a selective pressure, and only the best adapted clones are able to survive, due to the acquired genetic material and accumulated mutations on their genome. Also, these clones contain resistance and virulence genes which can be horizontally transferred and disseminated among patients, causing hospitals outbreaks.
P. aeruginosa resistance mechanisms
- Intrinsic resistance mechanisms
On the one hand, intrinsic resistance P. aeruginosa is based on some fundamental aspects:
the first one is ampC hyperexpression which provides resistance to aminopenicillins and most cephalosporins. Second one is MexAB-OprM efflux pump overexpression, which confers basal susceptibility to almost all beta-lactams (except imipenem) and fluoroquinolones. Furthermore, another intrinsic resistance mechanism is OprD porin inactivation, which frequently is associated with ampC hyperexpression, conferring resistance to all beta-lactams (Oliver et al., 2015).
As it has been mentioned, resistance to imipenem is mediated by the combination of OprD porin loss plus ampC hyperexpression. Thus, it is important to highlight the link of AmpC cephalosporinase to the cell wall recycling pathways, and resistance to beta-lactams is due to mutations in those pathways’ components (Pérez-Gallego et al., 2016).
Mutational overexpression of ampC is a usual cause of resistance to penicillins and most cephalosporins. A vast set of genes are related in the ampC regulation (see Figure 1). To
start, ampG encodes an inner membrane permease for GlcNAc-1,6- anhydromuropeptides, which in cytosol are processed by NagZ amidase. While bacteria are growing, anhydromuropeptides are processed by AmpD, which is also an amidase, by this way ampC induction is prevented (Lee et al., 2009). Contrarily, when bacteria are growing in presence of beta-lactams inducers, such as imipenem, large quantity of muropeptides are accumulated in the cytoplasm, leading to AmpR-mediated induction of ampC expression (Pérez-Gallego et al., 2016). The importance of AmpC in dynamics of resistance mechanisms development in P. aeruginosa when it is treated with imipenem and imipenem/relebactam fall on the ability of relebactam to avoid ampC hyperexpression caused by imipenem presence in outer membrane.
Figure 1: representation of P. aeruginosa ampC cephalosporinase regulation and peptidoglycan recycling under different conditions: a (inducer absence), b (inducer), c (non-inducer presence) (Moya et al., 2009).
The physiological role of OprD in P. aeruginosa is the transport of basic amino acids (Tamber and Hancock, 2006) and also it is the main porin used in P. aeruginosa for carbapenem diffusion (Fukuola et al., 1993). As it has been previously mentioned, due to P. aeruginosa intrinsic and acquired resistance mechanisms, only restricted classes of antibiotics can be used in antimicrobial therapy. Carbapenems such as imipenem are a very important antibiotics for therapy; nevertheless carbapenem-resistant P. aeruginosa strains are gradually increasing worldwide. So, lack or defective OprD confers P.
aeruginosa a basal level of resistance to carbapenems, such as imipenem (Li et al., 2012).
Efflux pumps are another intrinsic resistance mechanism for P. aeruginosa. Four multicomponent Multidrug-Resistant (MDR) Resistance Nodulation Division (RND)
efflux pumps have been described in P. aeruginosa: MexAB-OprM (Poole et al., 1993), MexCD-OprJ (Poole et al., 1996), MexEF-OprN (Köhler et al., 1997) and MexXY-OprM (Aires et al., 1999). These pumps have overlapping substrate spectra, but only MexAB- OprM is expressed at a sufficient level to confer intrinsic MDR in wild-type P. aeruginosa (Gotoh et al., 1994).
- Acquired resistance mechanisms
On the other hand, P. aeruginosa acquired resistance mechanisms are based on several attributes, for example: mutations in peptidoglycan-recycling genes provide resistance to antipseudomonal penicillins, cephalosporins and monobactams, and also changes in LPS cause resistance to polymyxins (Moya et al., 2009). Furthermore, a great rate of resistance is caused by efflux pumps, inasmuch as at least one pump overexpression causes resistance (Cabot et al., 2011). By way of illustration: MexAB-OprM overexpression plus OprD inactivation cause clinical resistance to meropenem and MexXY overexpression causes cefepime resistance in clinical isolates. Another acquired resistance mechanisms are caused by mutations in gyrA/gyrB and parC/parE genes, causing resistance to fluoroquinolones and also in ParRS two component system in which mutations cause colistin resistance (Bruchmann et al., 2013).
The frequency and spectrum of P. aeruginosa antibiotic resistance has increased worldwide. This spread has been attributed to the acquisition of resistance-encoding genetic material which is transferred between isolates via plasmids, integrons and transposons. Thereby, another P. aeruginosa acquired resistance mechanism rise from its ability to transfer horizontally resistance genes, for example: class B beta-lactamases (MBLs) are the most frequent cause of antibiotic resistance in P. aeruginosa. Extended spectrum beta-lactamases (ESBLs) and carbapenemases are found on type 1 integrons causing resistance to aminoglycosides. Therefore, aminoglycosides resistance can be horizontally transferred by aminoglycoside acetyltransferases such as aac(3’) causing gentamicin resistance, aac(6’) which causes tobramycin and amikacin resistance, and finally ant(2’)-l conditioning gentamicin and tobramycin resistance (Poole et al., 2011).
P. aeruginosa in vitro treatment
The prevalence of P. aeruginosa with MDR phenotypes is significant and increasing worldwide. MDR phenotypes compromise the selection of antimicrobial therapies delaying the administration of accurate antimicrobial therapies. This prevalence is one of the main causes for new antimicrobials development. Apart from classical antibiotics, during the last decades research has been focused on new beta-lactamase inhibitors, such as relebactam, which is a novel diazabicyclooctane (DBO) non-beta-lactam, class A and C (Ambler classification) beta-lactamase inhibitor (BLI). Older BLIs were not able to inhibit class C beta-lactamases such AmpC, because of this reason relebactam is a potential candidate to combine with imipenem, which is an ampC inducer (Zhanel et al., 2017).
Relebactam actually is on clinical trials being combined with imipenem to fight against infections caused by K. pneumoniae and P. aeruginosa. At a concentration of 4 µg/mL, relebactam also known as MK-7655 is able to reduce imipenem MICs for P. aeruginosa isolates and Enterobacteriaceae, with carbapenem resistance mediated by OprD loss (Olsen, 2015).
The aim of this work is to study the performance of relebactam when combined with imipenem. It is important to introduce that imipenem is an antipseudomonal carbapenem which inhibits the cross-linking of peptidoglycan, being able to enter into periplasmic space through outer membrane proteins (OMPs), so the bacteria break up and die.
Carbapenems such as imipenem remain the most important and most secure class of antibiotics currently available for the treatment of patients with serious Gram-negative infections, being used as agents of last resort. Thus, imipenem/relebactam seems to be an important treatment option for patients with carbapenem resistance and/or P. aeruginosa MDR phenotypes which are non-susceptible to available antipseudomonal agents.
P. aeruginosa difficulties remain on its resistance to imipenem which is present in approximately 30% of hospital isolates due to the ampC hyperexpression plus OprD porin loss (Zhanel et al., 2017). To avoid imipenem resistance during P. aeruginosa infections antibiotic treatments it is combined with relebactam. It has been tested by Karlowsky et al. (2019) that relebactam is able to restore imipenem susceptibility in clinical isolates
when co-administered, but it has no activity against bacteria by itself. Furthermore, to illustrate the potential of imipenem/relebactam it has been demonstrated by Lob et al.
(2019) that susceptibility rates of MDR phenotypes P. aeruginosa clinical isolates have been higher when treated in vitro with imipenem/relebactam in comparison with imipenem alone (92,4% vs 69,4%, respectively).
Imipenem/relebactam mechanism of action
There is not much literature about relebactam mechanism of action. Nevertheless, relebactam is expected to have a mechanism of action similar to avibactam, because of their structural similarities (Figure 2), except for an additional piperidine ring (Zhanel et al., 2017). Relebactam immediately acylates beta-lactamases and gradually de-acylates from them, producing a regenerated enzyme and an active inhibitor able to rebind.
Acylation takes place within the beta-lactamase active site serine residue and the carbonyl at position 7 in the cyclic urea core of relebactam (Olsen, 2015). Thus, the addition of relebactam prevent degradation of imipenem by certain class A and C beta-lactamases.
Then imipenem is able to cause its function by inhibiting peptidoglycan cross-linking.
Consequently, those bacteria that produce carbapenemases outside the inhibition spectrum of relebactam will continue being resistant to imipenem. Furthermore, relebactam is not affected by P. aeruginosa efflux pumps, realizing this compound as the best inhibitor to be used in combination with imipenem (Lob, 2019).
Imipenem/relebactam treatment
- Pharmacokinetics and pharmacodynamics
Pharmacokinetics is referred to a set of processes a drug is submitted though the body when it is administered. The pharmacokinetics of intravenous imipenem has been well established. Imipenem has a volume of distribution of 0.23-0.31 L/kg, with 20% of
Figure 1: relebactam (A) and avibactam (B) chemical structure similarities (Zhanel G. G. et al, 2017).
A B
protein binding, 60-70% of renal clearance and half-time elimination of 1h (Zhanel et al., 2017). Thus, imipenem/relebactam combination has been described by a two- compartment, linear model with first order elimination based on data from three phase I and one phase II trials (Zhanel et al., 2017). Mean volume of distribution was 12 L in females and 15.35 L in males (Butterton et al., 2010). Also, as imipenem, relebactam is approximately 20% binding protein (Butterton et al., 2010). Renal clearance and half- time elimination for relebactam reached from 5.3 to 9.1 L/h (Butterton et al., 2010). More studies are needed to standardize pharmacokinetics for relebactam results.
Pharmacodynamics is the set of physiological and biochemical drug effects, action mechanism, and also the relation between the drug concentration and its effect on the body. The pharmacodynamics of imipenem alone and imipenem/relebactam combination was tested in a murine thigh model by Mavridou et al. (2015). In this study, the effect of relebactam was not related with the maximum concentration of drug in serum. The T>MIC and area under the concentration-time curve AUC/MIC resulted comparable but did not appeared to be relation of effect with dose frequency. It was demonstrated that the relebactam AUC needed for a static effect was dependent on the dose of imipenem and the Minimum Inhibitory Concentration (MIC) of each strain (Mavridou et al., 2015).
- Animal studies
Imipenem has been deeply used in humans as agent of last resort. Imipenem/relebactam combination needed to be tested, then Powles et al. (2010) realized an in vivo efficacy study of relebactam in combination with imipenem in murine models of P. aeruginosa and Klebsiella pneumoniae infection. Mostly P. aeruginosa Colony-forming unit (CFU) rates animal infection models were reduced when imipenem was combined with relebactam in comparison with imipenem alone (Powles et al., 2010). These results encouraged the hypothesis of the present study, in which relebactam is expected to reduce resistance development to imipenem.
- Clinical trials
Currently MK-7655 is in two phase III clinical trials in combination with imipenem and cilastatin, for the treatment of hospital-acquired and ventilator-associated bacterial
pneumonia (HABP and VABP) caused by imipenem-resistant bacteria (NCT02452047 and NCT02493764). Furthermore, two completed phase II clinical trials have described the efficacy and the safety of imipenem/relebactam in treatment of complicated intra- abdominal infections (cIAI), complicated urinary tract infection (cUTI) and acute pyelonephritis (AP) compared to imipenem treatment alone (NCT01506271 and NCT01505634) (Zhanel et al., 2017).
- Place of imipenem/relebactam in therapy
Potential roles in therapy for imipenem/relebactam include treatment of suspected or documented infections caused by resistant Gram-negative bacilli-producing type A and C beta-lactamases. The use of these agents in patients with carbapenem-resistant Enterobacteriaceae infections will become the standard of care. The activity of imipenem/relebactam might be clinically useful in patients with suspected or reported infections caused by MDR P. aeruginosa (Zhanel et al., 2017).
HYPOTHESIS
Relebactam is expected to reduce resistance development to imipenem and the potential mechanisms of imipenem/relebactam resistance may show reduced fitness and virulence.
However, the potential mechanisms of resistance to imipenem/relebactam remain unknown.
OBJECTIVES
Below the main objectives of the present project are mentioned:
1. The first one is the study of resistance development in PAO1 and hypermutator phenotype of P. aeruginosa PAOMS when they were treated in vitro with imipenem and imipenem/relebactam at 4µg/mL fixed concentration.
2. The second objective is the in-silico analysis of the mutations caused by the imipenem pressure and imipenem when co-administered with relebactam.
3. The third objective is the study of the fitness strains through phenotypic effect of those mutations in C. elegans as an invertebrate model.
MATERIALS AND METHODS Strains
P. aeruginosa PAO1 and P. aeruginosaΔmutS as PAOMS have been used in this project.
- PAO1
P. aeruginosa PAO1 is a ubiquitous and metabolically versatile opportunistic pathogen, also the most commonly used strain for research. PAO1 strain is a derivative of the original Australian PAO isolate, which has been distributed worldwide to laboratories and strain collections (Klockgether et al., 2010). PAO1 has been used in this project as a wild-type strain to normalize and compare MICs and resistance evolution with standardized results. Complete genomic sequence and further information about PAO1 can be found on http://www.pseudomonas.com/feature/show?id=110059.
- PAOMS
Mismatches occurring during DNA replication or homologous recombination are repaired by different pathways, such as MMR, as it has been previously mentioned. The inactivation of the MMR system leads to an increase in the mutation rate because of its inability to repair mismatches correctly. Thus, P. aeruginosaΔmutS is a hypermutable strain which become adapted to many extreme environments such as CF patients’ lungs, catheters, mechanical ventilation, etc. Moreover, it has been described the presence of high proportion of P. aeruginosa isolates (20%) with an increased mutation frequency (mutators) in CF patients’ lungs due to their ability to adapt (Oliver et al., 2002).
In vitro resistance development
Procedure:
- Day 0: preparation of initial culture of each strain (PAO1 and PAOMS), 10 mL of MHB plus 1 colony from pure culture (stored at -80ºC).
- Day 1: inoculation of 50 μL from initial culture in a new tube containing 5 mL of MHB. Also, measurement optical density at 600nm since it reached to 0,2 (exponential growth). MHB was used as reference. Then, 10 mL MHB new tubes containing 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 µg/mL of imipenem and imipenem/relebactam at 4 µg/mL fixed concentration were inoculated with 50 μL PAO1 and PAOMS strains at 106 CFU/mL concentration (D.O.600nm=0,2) (Table 1). The tubes were incubated for 24h at 180 rpm and 37ºC.
Table 1: components for in vitro resistance development essay.
- Day 2: tubes from the highest antibiotic concentration showing visible growth were re-inoculated 10 μL in tubes with 10 mL fresh MHB containing the same concentration of antibiotic and all the above concentrations up to 64x imipenem concentration. The same procedure was repeated for a total of 7 consecutive days.
- Mentioned highest antibiotic concentration tubes with visible growth corresponding to days 2 and 8 (mutants from days 1 and 7) were diluted to 106 CFU/mL and spread 100 μL in MH plates, then these plates were incubated ON at 37ºC. Two colonies of each plate were selected and stored frozen -80ºC for further studies.
0.125x 0.25x 0.5x 1x 2x 4x 8x 16x 32x 64x
MH 10mL 10mL 10mL 10mL 10mL 10mL 10mL 10mL 10mL 10mL
[IMI] 0.125 μg/mL
0.25 μg/mL
0.5 μg/mL
1 μg/mL
2 μg/mL
4 μg/mL
8 μg/mL
16 μg/mL
32 μg/mL
64 μg/mL
[REL] 4
μg/mL 4 μg/mL
4 μg/mL
4 μg/mL
4 μg/mL
4 μg/mL
4 μg/mL
4 μg/mL
4 μg/mL
4 μg/mL Inoculum 50 μL 50 μL 50 μL 50 μL 50 μL 50 μL 50 μL 50 μL 50 μL 50 μL
- Finally, mutants were obtained: 2 strains, 5 series per strain, 2 treatments per each strain and series, day 1 and 7 mutants per each treatment. These 40 mutants were duplicated, concluding with 80 mutants.
Susceptibility testing
MICs have been determined by two different protocols. Both have been used to determinate imipenem and imipenem/relebactam MICs, and also further antibiotics.
Obtained MICs helped among others to advance genomic reorders as SNPs, Insertions- Deletions (InDels) or premature stop codons in P. aeruginosa resistome genes. Both together these protocols have been extensively used by our Group (Del Barrio-Tofiño et al., 2019).
- Sensititre ® custom plates
To determine MICs of ticarcillin (TIC), ciprofloxacin (CIP), piperacillin/tazobactam (P/T), aztreonam (AZT), ceftazidime (TAZ), ceftolozane/tazobactam (C/T), ceftazidime/avibactam (CZA), cefepime (FEP), amikacin (AMI), tobramycin (TOB), imipenem (IMI), meropenem (MERO) and colistin (COL) broth microdilution protocol from Sensititre custom plates by Thermo Fisher Scientific ® has been used.
Procedure:
- Inoculation: all broths were at room temperature before use.
- Colony selection: 3-5 colonies were picked up from fresh primary agar plate and were emulsified in sterile water and adjusted to a 0.5 McFarland Standard.
- Inoculum preparation: 10 μL of the suspension were transferred into a tube of cation adjusted MH broth with TES buffer to give an inoculum of 105 cfu/mL (Table 2).
- Inoculating a plate: 50 μL were transferred to containing antibiotics plate (Table 2). Then, the plate was covered with an adhesive seal.
- Incubation: 34-36ºC for 18-24h.
Table 2: organism, suspension, inoculums and media for MICs determination.
- MHB microdilution
The second protocol for the study of imipenem and imipenem/relebactam MICs was based on MHB microdilution. In this case each MIC of imipenem and imipenem/relebactam was studied in two different plates. On the first one, there was a decreasing imipenem concentration range, starting at 64 µg/mL and ending at 0.125 µg/mL. On the second plate, there was also the same decreasing imipenem concentration range accompanied by relebactam at 4 µg/mL fixed concentration. 105 CFU/mL bacterial concentration (0.5 McFarland Standard) was necessary to plates inoculation.
This protocol played an important role on the in vitro dynamics study of relebactam as a new beta-lactamase inhibitor, and how its selective pressure affected to PAO1 and PAOMS.
Characterization of resistance mechanisms
- Library preparation and whole genome sequencing (WGS)
Following protocols have been necessary for PAO1 and PAOMS whole-genome sequencing (Cabot et al., 2016; López-Causapé et al., 2017).
First step was bacteria DNA isolation. This protocol corresponded to High Pure PCR Template Preparation Kit Roche Diagnostics®. DsDNA quality was analyzed by NanoDrop™ 2000 Thermo SCIENTIFIC®. Absorbance at 260/230 nm had to range between 1.9 to 2.1 and 1.7 to 1.9 at 260/280 nm to avoid spots. Just 1 μL of dsDNA of each sample was used.
Organism McFarland suspension
Final inoculum
Inoculum transfer
Broth Plate
reconstitution
P. aeruginosa Water 105 cfu/mL 10 μL MHB 50 μL
When dsDNA parameters were in range, DNA quantification began with Quant-iT™
dsDNA Assay Kit by Thermo SCIENTIFIC®. Then, DNA Library preparation was started when dsDNA reached 0.2 ng/μL. The protocol used in this experiment corresponded to Nextera XT Library Prep Kit Illumina ®.
Workflow:
1. Tagment Genomic DNA: a transposome was used to tagment gDNA. This process fragmented DNA and then tagged DNA with adapter sequences.
2. Amplify Libraries: tagmented DNA was amplified using a limited-cycle PCR program, which added barcodes to DNA. The index adapters and Nextera PCR Master Mix were added directly to the tagmented gDNA.
3. Clean Up Libraries: magnetic beads were used to purify the DNA library and removed short fragments.
4. Normalize Libraries: library quantification was normalized to ensure more equal library representation in the pool library.
5. Pool Libraries: pooling libraries combined equal volumes of normalized libraries in a single tube.
6. Denature Libraries: library pool was diluted and heat-denatured before loading libraries for the sequencing run.
7. Sequencing: paired-end libraries were denatured and pool libraries were sequenced on an Illumina MiSeq ® benchtop sequencer with MiSeq reagent kit Illumina Inc., resulting in 500 bp paired-end reads.
- Variant calling
Variant calling analysis was performed as described has been previously by our Group Cabot et al. (2016) and López-Causapé et al. (2017). Briefly, after sequencing on a MiSeq benchtop sequencer (approximately 72h), 2 fastq files corresponding to forward and reverse sequencing were generated from each mutant. Paired-read samples were then aligned to the PAO1 reference genome (downloaded from:
http://pseudomonas.com/strain/show/107) with Bowtie2 v2.2.4 software, which exported fastq files to SAM (Sequence Alignment Map) files. After, SAMtools v0.1.16 was used to convert SAM into BAM (Binary Alignment Map) files to obtain a total pile up and raw file from each mutant. Therefore, SNPs (Single Nucleotide Polymorphisms) and InDels
screening was realized lying on the same quality parameters as López-Causapé et al.
(2017). After parameters validation, file format conversion from a raw file to a variant calling file (VCF) was facilitated by ANNOVAR software. To conclude, variant calling analysis SnpEff v4.2 software was used to annotate SNPs and InDels in a final accurate file (results are shown of Tables 3a and 3b).
C. elegans killing assay
The assay for studying bacterial killing of C. elegans was performed as described previously by Sánchez-Diener et al. (2017). Briefly, a fresh culture of each strain was layered onto a plate containing 5 ml of potato dextrose agar (PDA). After spreading the bacterial culture, the plates were incubated overnight at 37°C. Then, 5 worms per plate were laid on the bacterial lawn. The plates were incubated at 24°C and scored at 24 h, 48h, 72 h, 144h and 168 h to detect the presence of living worms. The nematodes were examined at ×20 and ×40 magnification. Three independent replicate experiments per bacterial strain were performed and means and standard deviations (SDs) were recorded.
C. elegans virulence score (CEVS) was developed for the first time by Sánchez-Diener et al. (2017). Therefore, the isolates were classified into 5 virulence levels depending on the effect on the growth of the nematodes. In CEVS 1 and 2, the strains were considered as nonvirulent if did not kill the nematode at 168h. CEVS 3 was considered when the number of live nematodes at 168 h reached from 1 to 5. CEVS 4 was considered when the number of live nematodes at 168 h was just 1 or less. Finally, CEVS 5 was considered when the number of live nematodes recorded at 72 h was 0.
RESULTS
Imipenem/relebactam combination has variable activity against P. aeruginosa isolates.
This activity is thought to be due to inhibition of low-level imipenem hydrolysis carried out by P. aeruginosa AmpC endogenous enzyme. So, as it has been previously mentioned, imipenem/relebactam combination remains under evaluation. Furthermore, imipenem/relebactam is not currently FDA-cleared for clinical use. The European Society of Clinical Microbiology and Infectious Diseases (EUCAST) has recently determined new interpretations about antimicrobial susceptibility testing for P. aeruginosa. Then,
according to Clinical Breakpoint Tables v. 9.0, valid from 2019-01-01, P. aeruginosa is considered as imipenem resistant if MIC breakpoint reaches >4 mg/L. Lower values are considered as sensitive.
Dynamics of resistance development to imipenem and imipenem/relebactam
At the present work, in vitro dynamics resistance development to imipenem has been studied for PAO1 and PAOMS for 7 days under two different pressures: imipenem and imipenem/relebactam at 4 µg/mL fixed concentration. The development of high-level imipenem resistance was fastest for PAOMS when treated with imipenem alone, reaching imipenem maximum MIC of 64 µg/mL at 5th day, in contrast to PAO1 just arrived at 16 µg/mL at 7th day (Figure 3.a). Furthermore, when both strains were treated with imipenem/relebactam, PAOMS also reached at imipenem maximum MIC at 5th day while PAO1 just reached 4 µg/mL MIC at 7th day (Figure 3.b). PAO1 when treated with imipenem reached its maximum at 16 µg/mL MIC at 7th day (Figure 3.a), but when it was treated with imipenem/relebactam just arrived 4 µg/mL MIC at 7th day (Figure 3.b).
Comparison graphics of PAO1 are shown on Figure 4.a, where resistance levels to imipenem are lower when PAO1 was treated with imipenem/relebactam, in comparison with treated with imipenem alone. Same procedure was realized for PAOMS, in this case when treated with imipenem, MIC reached 64 µg/mL at the 5th day and when treated with imipenem/relebactam arrived 64 µg/mL at for imipenem at 7th (Figure 4.b). These results suggested that the previously mentioned ability of relebactam may be interfered by accumulated mutations in PAOMS.
Figure 3.a: in vitro resistance development to imipenem in PAO1 (green) and PAOMS (red) for 7 consecutive days when treated with imipenem alone.
Figure 3.b: in vitro resistance development to imipenem in PAO1 (blue) and PAOMS (purple) for 7 consecutive days when treated with imipenem/relebactam.
Inverse correlation between resistance and virulence in the C. elegans model
As it has been previously documented by Sánchez-Diener et al. (2017) there is an opposite association between antimicrobial resistance and virulence in the C. elegans infection model. This nematode has been commonly used as a host due to its characterization and simplicity. Furthermore, this infection model has some advantages, such as being low cost, manageable, large-scale screening, and it does not raise ethical concerns for drug testing in early stages. In the present work, virulence and lethality of 5 PAO1 mutants treated with imipenem, 5 with imipenem/relebactam, 5 PAOMS mutants treated with imipenem and 5 with imipenem/relebactam were tested in C. elegans model. In these experiments, Escherichia coli OP50, which is an optimum food source for C. elegans, was used as a positive control, and wild-type PAO1 and PAOMS, as a negative control.
Weak virulence differences between these two strains when treated with imipenem alone could be clearly obtained in C. elegans model (Figure 5.a and Figure 5.b). When both strains were treated with imipenem/relebactam, PAO1 resulted more virulent than PAOMS as can be seen on Figure 5.b, while PAOMS in vitro treated with imipenem/relebactam was not able to kill the nematodes, being considered as non- virulent. As it has previously commented, strains can be classified in 5 virulence levels depending on the effect on the growth of the nematodes (Sánchez-Diener et al., 2017), then looking at Figure 5.a and 5.b, most of PAOMS treated with imipenem/relebactam resulted in a CEVs 3, considered as an intermediate virulence. In contrast, most of PAO1 treated with imipenem/relebactam, resulted in a CEVs 4-5, being considered as virulent.
When PAO1 and PAOMS were treated with imipenem alone, trend was to be more virulent, being considered as a CEVs 4-5.
Figure 4.a: in vitro resistance development to imipenem for PAO1 when treated with imipenem alone (green) and imipenem/relebactam (purple) for 7 consecutive days.
Figure 4.b: in vitro resistance development to imipenem for PAOMS when treated with imipenem alone (red) and imipenem/relebactam (blue) for 7 consecutive days.
Mutants characterization by WGS
Recently introduction of WGS technology is advancing a new dimension for MDR pathogens and mutational resistome. The expression “mutational resistome” has been recently put into practice for the collection of mutations related to antibiotic resistance levels in absence of horizontal gene transfer (HGT) (Cabot et al., 2016; López-Causapé et al., 2017). Tables 3.a and 3.b summarize MICs for several antibiotics and mutations causing resistance in P. aeruginosa when treated with imipenem and imipenem/relebactam at 4 µg/mL fixed concentration. When connecting treatment and MICs, resistance to imipenem seemed to be related with mutations in ampC regulation genes (mutations in ampDh3, ampR, ampC and ampD) and oprD with premature codon stop. Moreover, resistance to imipenem/relebactam required OprD loss and MexAB- OprM overexpression (mutations in mexB, mexR, nalC and nalD).
Figure 5.a: lethality of PAO1 in the C.elegans model.
The mean numbers of surviving nematodes at 0, 24, 72, and 168 h are shown. PAO1 (black continuous line) and E. coli OP50 (black discontinuous line) are included for comparative purposes.
Figure 5.b: lethality of PAOMS in the C.elegans model.
The mean numbers of surviving nematodes at 0, 24, 72, and 168 h are shown. PAOMS (black continuous line) and E. coli OP50 (black discontinuous line) are included for comparative purposes.
PAO132≤442≤0.522≤0.12≤2≤0.254121≤0.12 PAOMS648821440.254142220.25 m132≤442≤0.512≤0.12≤2≤0.258-164180.5-1oprD(nt1205InsC) m232≤442≤0.512≤0.12≤2≤0.25162-4180.5-1oprD(W339*) m132≤442≤0.512≤0.12≤2≤0.258-162-4140.5-1 m232≤442≤0.512≤0.12≤2≤0.258-162-4140.5-1 m132≤442≤0.522≤0.12≤2≤0.258-164180.5-1oprD(V147F), mexT(nt900InsCTCGCGGATCA) m232≤442≤0.522≤0.12≤2≤0.2516-322-4180.5-1oprD(V147F) m132≤442≤0.512≤0.12≤2≤0.25162-4180.5oprD(L292*), mexT(G300C) m232≤442≤0.51≤1≤0.12≤2≤0.2516-322180.5oprD(L292*), mexT(G300C) m132≤442≤0.51≤1≤0.12≤2≤0.258-162120.5 m232≤442≤0.522≤0.12≤2≤0.2582120.5 m1328441240.2540.516-32428-160.5oprD(Q327*), mrcB(L546P) m2328421240.2540.53242160.5oprD(nt415Δ1), mrcB(L546P) m1648821440.2541324280.5oprD(W277*), dacC(K283R), mrcB(L546P) m2648441440.254132428-160.5oprD(nt630InsG), mexT( E330D; nt986InsCT), dacC(K283R) m1648841240.254116-32428-160.5oprD(W277*), mexT(G300C; E330D; W332R; W332C), mrcB(L546P) m232882122≤0.1241324280.5oprD(S278P) m1128163281480.2541328280.5oprD(W277*), mrcB(L546P) m2128161641480.258116-32828-160.5oprD(W277*), mrcB(L546P) m16416882480.2581328280.5oprD(W277*), mrcB(L546P) m26481681440.58116-328280.5oprD(W277*), mrcB(L546P) m132≤442≤0.512≤0.12≤2≤0.2516-322-4180.5oprD(W339*), mexT(E330D; W332R) m232≤442≤0.512≤0.12≤2≤0.258-162-4180.5oprD(W339*), mexT(E330D; W332R) m132≤442≤0.512≤0.12≤2≤0.258-162-4110.25-0.5 m232≤442≤0.512≤0.12≤2≤0.258-16214-80.5 m132≤442≤0.512≤0.12≤2≤0.258-162180.5oprD(nt909Δ1) m232≤442≤0.512≤0.12≤2≤0.258-16210,50.5 m132≤442≤0.512≤0.12≤2≤0.258-162120.25-0.5 m232≤442≤0.512≤0.12≤2≤0.258-162120.25mexT(nt900InsCTCGCGGATCA) m132≤442≤0.512≤0.12≤2≤0.258-162140.25-0.5mexT(E330D; W332R) m232≤442≤0.522≤0.12≤2≤0.258-162-4140.5oprD(G106D), mexT( E330D; W332R) m164884124≤0.1280.53282160.5oprD(W339*), phoQ(H214Y), mrcB(L546P) m264161681440.2540.5328216-320.5-1oprD(W339*), phoQ(H214Y), mrcB(L546P) m1648841440.2540.5324280.25-0.5oprD(nt101InsG), mexT( E330D; nt983InsCCCAGGC), dacC(K283R), mrcB(L546P) m26416881240.2540.516-324280.5oprD(W138*), mexT(E330D), dacC(K283R) m16481641440.254116-328280.5oprD(nt415Δ1), mrcB(L546P) m26481682240.258132828-160.5oprD(nt434Δ1), mrcB(L546P) m16464882440.2581328280.5oprD(nt181Δ1), mexT(E330D; W332R), mrcB(L546P) m2128321682440.258116-328280.5oprD(M1I), mexT( E330D; W332R), mrcB(L546P) m112832881440.2581328280.5oprD(W138*), mrcB(L546P) m2128321641480.258116-328280.5oprD(W277*), mrcB(L546P)
MICa TICP/TZAZTTAZC/TCZAFEPCIPAMITOBIMIMERCOLIPIP+REL (4µg/mL) IMID1
P1 P2 P3 P4 P5 M1 M2 M3 M4 M5 IMI/RELD1
P1 P2 P3 P4 P5 M1 M2 M3 M4 M5
Mutations
Table 3.a: ticarcillin (TIC), ciprofloxacin (CIP), piperacillin/tazobactam (P/T), aztreonam (AZT), ceftazidime (TAZ), ceftolozane/tazobactam (C/T), ceftazidime/avibactam (CZA), cefepime (FEP), amikacin (AMI), tobramycin (TOB), imipenem (IMI), meropenem (MERO) and colistin (COL) MICs and also imipenem (IP) and imipenem/relebactam (IP+REL) MICs from MHB microdilution. The main genes and mutations known to increase resistance to imipenem for
