Departamento de

Microbiología II

Universidad

Complutense. Madrid

Hospital Universitario Ramón y CajalSERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA

Dr. Rafael Cantón

Resistance to new β-lactam-

β-lactamase inhibitor

combinations: a new step in

the muti-drug resistance?

Disclosures

▪ Participation in educational programs

- Angelini

- Beckman

- MSD

- Pfizer

- Roche

- ThemoFisher

- Zambon

▪ Participation in research studies- AstraZeneca

- BioMerieux

- Cepheid

- MSD

▪ Evaluation of clinical trials

- Bayer

- MSD

▪ Why new β-lactam-β-lactamase inhibitor combinations?

▪ Which are the new β-lactam-β-lactamase inhibitor combinations?

▪ How the new β-lactam-β-lactamase inhibitor combinations work?

▪ How is the resistant development? How I can detect it?

▪ How can avoid emergence of resistance?

Antibacterial agents in clinical

development: an analysis of the

antibacterial clinical development

pipeline, including tuberculosis.

Geneva: World Health Organization;

2017 (WHO/EMP/IAU/2017.11).

Prioritization of

pathogens to guide

research and

development of

new antibiotics

Most frequent microorganisms involved in HAI

and antimicrobial resistance (ICUs in EU, 2015)

%

3rd gen. cephalosporin R

20% E. coli

42% Enterobacter spp.

43% Klebsiella spp.

Carbapenem R

11% Klebsiella spp.

24% P. aeruginosa

69% A. baumannii

Mayor problems of

resistance (R)

ECDC. Healthcare-associated infections acquired in intensive care units. In: ECDC. Annual epidemiological report for 2015. Stockholm: ECDC; 2017.

Top-10 pathogens in nosocomial ICU infections in EU

3rd gen. cephalosporin resistance worldwide

Overall trend in EU region (ATLAS surveillance study, 2016)

Escherichia coli Klebsiella pneumoniae

ATLAS surveillance. Available at: https://atlas-surveillance.com/#/heatmap/resistance. [Accessed April 2018]

Spain: 20.8% Spain: 38.4%

3rd gen. cephalosporin resistance in Europe

(invasive isolates, EARS-net 2016)

Escherichia coli Klebsiella pneumoniae

Antimicrobial resistance surveillance in Europe 2016. Annual Report of the European Antimicrobial Resistance Surveillance

Network (EARS-Net). Stockholm: ECDC; 2017. Available at: https://ecdc.europa.eu/en/publications-data/antimicrobial-

resistance-surveillance-europe-2016. [Accessed April 2018].

15.0% 22.3%

Klebsiella pneumoniae

Carbapenem (meropenem) resistance worldwideOverall trend in EU region (Atlas surveillance study, 2016)

ATLAS surveillance. Available at: https://atlas-surveillance.com/#/heatmap/resistance. [Accessed April 2018].

Escherichia coli

Spain: 0% Spain: 2.2%

Carbapenem (meropenem) resistance in Europe

(invasive isolates, EARS-net 2016)

Escherichia coli Klebsiella pneumoniae

Antimicrobial resistance surveillance in Europe 2016. Annual Report of the European Antimicrobial Resistance Surveillance

Network (EARS-Net). Stockholm: ECDC; 2017. Available at: https://ecdc.europa.eu/en/publications-data/antimicrobial-

resistance-surveillance-europe-2016. [Accessed April 2018].

0.1% 2.1%

Carbapenemase producing Enterobacteriaceae in Europe

0 10 20 30 40 50 60 70 80 90 100

Austria

Belgium

France

Germany

Greece

Hungary

Israel

Italy

Montenegro

Portugal

Romania

Serbia

Spain

Turkey

UK

KPC NDM VIM OXA-48-Like Other R-mechanism

EuSCAPE program (Nov 1, 2013 - April 30, 2014. ECDC)

Type of carbapenemase in K. pneumoniae by country (only those with ≥10 isolates)

Data extracted from: Grundmann et al. Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European

survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE): a prospective, multinational study. Lancet Infect Dis. 2017; 17:153-63.

Carbapenem and colistin resistance in K. pneumoniae

in Europe (invasive isolates, EARS-net 2016)

Summary of the latest data on antibiotic resistance in the European Union EARS-Net surveillance data. November 2017

https://ecdc.europa.eu/sites/portal/files/documents/EAAD%20EARS-Net%20summary.pdf

Colistin-R carbapenemase

producing K. pneumoniae

clones (ST11, ST101, and

ST512) disseminated in

Italy*, are also present in

Spain**

*Monaco et al. Euro Surveill 2014;19(42)

pii: 20939

** Oteo et al. J Antimicrob Chemother.

2016; 71:3392-9.

Meropenem

Antimicrobial resistance worldwide in P. aeruginosa

Overall trend in EU region (Atlas surveillance study, 2016)

ATLAS surveillance. Available at: https://atlas-surveillance.com/#/heatmap/resistance. [Accessed April 2018].

Ceftazidime

Spain: 25.7% Spain: 18.5%

Antimicrobial resistance in P. aeruginosa in

Europe (invasive isolates, EARS-net 2016)

% of invasive isolates with combined resistance to ≥3 antimicrobials* in 2016

(*piper/tazob, ceftazidime, fluoroquinolones, aminoglycosides and/or carbapenems)

Antimicrobial resistance surveillance in Europe 2016. Annual Report of the European Antimicrobial Resistance Surveillance Network (EARS-Net). Stockholm:

ECDC; 2017. Available at: https://ecdc.europa.eu/en/publications-data/antimicrobial-resistance-surveillance-europe-2016. [Accessed April 2018].

▪ Why new β-lactam-β-lactamase inhibitor combinations?

▪ Which are the new β-lactam-β-lactamase inhibitor combinations?

▪ How the new β-lactam-β-lactamase inhibitor combinations work?

▪ How is the resistant development? How I can detect it?

▪ How can avoid emergence of resistance?

0

2

4

6

8

10

12

14

16

18

1983-1987 1988-1992 1993-1997 1998-2002 2003-2007 2008-2012 2013-2017 2018-

nu

mb

er

of

ne

w a

nti

mic

roib

ials

Years

Ceftobiprole (2013)

Telavancin (2014)

Dalbavancin (2015)

Tedizolid (2015)

Oritavancin (2015)

Ceftolozane-tazobactam (2015)

Ceftazidime-avibactam (2016)

Delafloxacin (2017)

Ozenoxacin (2017)

Meropenem-vaborbactam (2017)

Antibacterial agents approved by FDA and/or EMA

Ceftaroline (2010)

Fidaxomicin (2012)

Delamanid (2012)

Bedaquiline (2012)

Updated from Shlaes et al. AACh 2013, 57:4605-7

Plazomicin (2018)

Eravacycline (2018)

Antibacterial drug FDA / EMA approved for…

Ceftaroline CABP, ABSSSI (incl. MRSA)

Fidaxomicin CDI

Bedaquiline, Delamanid Pulmonary MDR tuberculosis

Telavancin NP due to S. aureus (limited to MRSA in EU)

Ceftobiprole HABP (not VABP), CABP

Dalbavacin ABSSSI

Oritavancin ABSSSI due to Gram-(+)s including MRSA

Tedizolid ABSSSI

Ceftolozane-tazobactam cUTI (incl. acute pyelonephritis), cIAI (+metronidazole)

Ceftazidime-avibactam cUTI (incl. acute pyelonephritis), cIAI (+metronidazole), HABP incl.

VABP, infect. aerobic Gram-(-)s adults with limited options

Delafloxacin ABSSSI

Meropenem-vaborbactam cUTI (incl. acute pyelonephritis)

Ozenoxacin Impetigo due to S. aureus or S. pyogenes

Plazomicin

Eravacycline

ABSSSI: acute bacterial skin and soft tissue infections; cIAI: complicated intraabdominal infecions; CDI: C. difficile infection, cUTI: complicated

urinary tract infections; CABP: community acquired bacterial pneumonia; HABP: hospital acquired bacterial pneumonia; MDR: multi-drug

resistant; NP: nosocomia pneumonia; VABP: ventilator associated bacterial pneumonia

▪ Why new β-lactam-β-lactamase inhibitor combinations?

▪ Which are the new β-lactam-β-lactamase inhibitor combinations?

▪ How the new β-lactam-β-lactamase inhibitor combinations work?

▪ How is the resistant development? How I can detect it?

▪ How can avoid emergence of resistance?

Ceftazidime Avibactam

• 3rd gen. cephalosporin active against

Enterobacteriaceae and P. aeruginosa

• Affected by

- AmpC derepression

- ESBL (partial) and carbapenemases

- Porin or efflux pumps

• β-lactamase inhibitor with the highest

inhibitor spectrum

• Inhibitor of different β-lactamase classes

- A (ESBLs and KPCs)

- C (AmpC)

- D (ESBLs and OXA-48-type)

Approved for:

- Complicated urinary tract infection (including acute pyelonephritis)

- Complicated intra-abdominal infections (associated with metronidazole)

- Hospital-acquired pneumonia (including ventilator-associated pneumonia)

- Infections in adults due to aerobic Gram-negative infections with limited options

Sharma R, et al. Clin Ther 2016;38:431-44; Rodríguez-Baño J, et al. Clin Microbiol Rev 2018;31.pii: e00079–17; Ceftazidime-avibactam SPC.

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR-Product_Information/human/004027/WC500210234.pdf [Accessed April 2018]

Ehmann DE et al. Proc Natl Acad Sci. 2012;29:11663–11668; 3. Aktaş Z, et al. Int J Antimicrob Agents 2012;39:86-9.

Inhibited by avibactam

Serin-enzymes

A C D

Zinc enzymes

B

B-LACTAMASES

Active site

Nucleotidic

sequence

CARBAPENEMASES

(METALO-β-LACTAMASES

VIM, NDM, IMP, …)

ESBL (CTX-M)

CARBAPENEMASES

(KPC)

CARBAPENEMASES

(OXA-48)AmpC

Bush & Bradford. Cold Spring Harb Perspect Med. 2016; 6(8). pii: a025247

β-lactamase classification

CAZ, ceftazidime; CAZ–AVI, ceftazidime–avibactam

López-Hernández I, et al. Activity of ceftazidime-avibactam against multidrug-resistance Enterobacteriaceae expressing combined

mechanisms of resistance. Enferm Infecc Microbiol Clin 2017;35:499–504.

CAZ and CAZ–AVI* MIC distributions in ESBL and/or

AmpC producing K. pneumoniae isolates in Spain

*Ceftazidime–avibactam is the property of Pfizer. CAZ, ceftazidime; CAZ–AVI, ceftazidime–avibactam; KPC, K. pneumoniae carbapenemase;

MBL, metallo-β-lactamase OXA, oxacillinase.

Jonge BL, et al. In vitro susceptibility to ceftazidime-avibactam of carbapenem-nonsusceptible Enterobacteriaceae isolates collected during the

INFORM Global Surveillance Study (2012 to 2014). Antimicrob Agents Chemother. 2016;60:3163–9.

0

10

20

30

40

50

60

70

80

90

100

0,0

1

0,0

3

0,0

6

0,1

2

0,2

5

0,5 1 2 4 8

16 32 64

128

>12

8

CAZ CAZ-AVI

0

50

100

150

200

250

300

0,0

1

0,0

3

0,0

6

0,1

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16 32 64

128

>12

8

CAZ CAZ-AVI

0

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25

30

35

40

45

50

0,01

0,03

0,06

0,12

0,25 0,

5 1 2 4 8

16 32 64

128

>128

CAZ CAZ-AVI

0

20

40

60

80

100

120

140

0,01

0,03

0,06

0,12

0,25 0,

5 1 2 4 8

16 32 64

128

>128

CAZ CAZ-AVI

Carbapenemase-negative KPC-positive, MBL-negative

OXA-48-like positive, MBL-negative MBL positive

CAZ vs. CAZ–AVI* in carbapenem nonsusceptible

Enterobacteriaceae (INFORM Global Surveillance study, 2012-2014)

Torrens et al. Antimicrob Agents Chemother 2016; 60:6407-10

% of susceptible isolates

All isolates (n=190) AmpC hyperp. (n=46) Multi-drug-R (n=63)

CAZ CAZ-AVI MER CAZ CAZ-AVI MER CAZ CAZ-AVI MER

64.7 91.1 77.4 10.9 76.1 41.3 27.0 77.8 74.1

CAZ and CAZ–AVI* MIC distributions in

bacteriemic P. aeruginosa isolates in Spain

Ceftolozane Tazobactam

• 4th gen. cephalosporins derived from ceftazidime

(higher PBP affinity)

• High intrinsic activity against Pseudomonas

aeruginosa, including those with efflux/AmpC

hyperexpression and/or porin deficiency

• Partially affected by ESBLs and fully affected by

carbapenemases

• Suicide inhibitor

• Inhibitor of class A β-lactamases,

including ESBls but not KPCs

• Scarce inhibition of class C (AmpC)

and null of class D (OXAs)

Approved for: - complicated UTI (including acute pyelonephritis)

- complicated intraabdominal infection (associated with metronidazol)

Scott LJ. Drugs 2016; 76:231-42

Inhibited by avibactam

Serin-enzymes

A C D

Zinc enzymes

B

B-LACTAMASES

Active site

Nucleotidic

sequence

CARBAPENEMASES

(METALO-β-LACTAMASES

VIM, NDM, IMP, …)

ESBL (CTX-M)

CARBAPENEMASES

(KPC)

CARBAPENEMASES

(OXA-48)AmpC

Bush & Bradford. Cold Spring Harb Perspect Med. 2016; 6(8). pii: a025247

β-lactamase classification

Inhibited by tazobactam

▪ Specifically developed from ceftazidime to enhanced antibacterial activity against P. aeruginosa and to reduce neurotoxicity (seizures)

▪ The strategy to improve the anti-pseudomonal activity is based on:

▪ maintenance of high PBP affinity

▪ increased outer membrane permeability

▪ improved stability to AmpC β-lactamase

Toda A, et al.. Bioorg Med Chem Lett 2008;18:4849-52. Zhanel et al. Drugs 2014; 74:31-51

Dimethylacetic acid moiety

enhances antipseudomonal activity

Oxime confers

β-lactamase stability

2-aminoethylureido group: optimal balance of activity

against AmpC β-lactamase-producing P. aeruginosa

and weakest convulsion-inducing potential (pka=7.95)

2-methylpyrazole group was found to have

the best activity against P. aeruginosa

Pyrazole ring provides stability

against AmpC β-lactamase-

overproducing P. aeruginosa

Aminothiadizaole ring enhances

activity against Gram-(-) bacilli

Ceftolozane

Study (no. of P. aeruginosa)Ceftazidime-avibactam Ceftolozane-tazobactam

% S MIC50/MIC90 % S MIC50/MIC90

Buehrle AAC 2016 (n=38) 92% 1 / 8 92% 1 / 4

Gonzalez Ann Lab Med 2017 (n=45) 82% 2 / 16 87% 1 / 8

Grupper AAC 2017 (n=290) 81% 1 / 8 91% 1 / 4

Humphries AAC 2017 (n=309)* 62% ND 73% ND

Wi AAC 2018 (n=42)** 71.4% 8 / 16 95% 2 / 4

Sader JAC 2018 (n=423) 96.2% - 96.5 -

*Isolates included if resistant to >1 antipseudomonal β-lactam (ceftazidime, cefepime, meropenem,

imipenem or piperacillin-tazobactam)

Ceftazidime-avibactam vs. ceftolozane-tazobactam

in Pseudomonas aeruginosa

▪ Different studies document in vitro activity of both antimicrobials against

multi-drug resistant (MDR) P. aeruginosa isolates

▪ At least 6 studies evaluated the activity of both antimicrobials in parallel

against P. aeruginosa, including MDR populations

MIC distribution for ceftolozane-

tazobactam (C/T) (light gray) and

ceftazidime-avibactam (CZA)

(dark gray) in P. aeruginosa (n=309) isolates with β-lactam

resistance

▪ 72.5% C/T susceptible (≤4 mg/L)

▪ 61.8% CZA susceptible (≤8 mg/L)

▪ Among C/T resistant isolates,

9.1% were CZA susceptible,

whereas 36.4% of CZA-resistant

isolates were susceptible to C/T

Resistance profiles of P. aeruginosa

(n=309) that were resistant at least to

piperacillin-tazobactam, ceftazidime,

cefepime, imipenem or meropenem

In all situations, C/T susceptibility was higher that that of CZA

Int J Antimicrob Agents. 2015; 46:502-10

Antibiotic % S

C/T 94.4

CAZ 76.0

MER 64.6

P/T 63.6

IMP 60.0

LEV 61.6

▪ Why new β-lactam-β-lactamase inhibitor combinations?

▪ Which are the new β-lactam-β-lactamase inhibitor combinations?

▪ How the new β-lactam-β-lactamase inhibitor combinations work?

▪ How is the resistant development? How I can detect it?

▪ How can avoid emergence of resistance?

▪ MIC increase from 2-4 mg/L

to 32->256 mg/L in isolates

from three patients (8%, 3/37)

• Patients with resistant isolates

were treated during 15 days

(range 10-19 days)

▪ Mutations in the blaKPC

sequence after ceftazidime-

avibactam exposure

Emergence of CAZ-AVI acquired resistance

2015; 59:5324-30

Emergence of ceftazidime-avibactam acquired

resistance in Enterobacteriaceae

▪ Overexpression of extended-spectrum AmpC (ESAC) in E. cloacae

and mutations in AmpC from P. aeruginosaLahiri et al. JAC 2014; 69:2942-6; Lahiri et al. JAC 2015; 70:1650-8

▪ KPC-K. pneumoniae with multiple resistance mechanisms:

- KPC-3 overexpression plus porin deficiency (OmpK35/OmpK36),

and SHV-12 and enhance efflux activity (AcrAB)Nelson et al. AAC 2017; 61:e00989-17; Humphries et al. AAC 2017; 61:e00537-17

- Doble or triple blaKPC-3 mutationsShields et al, AAAC 2017; 61: e02097-16; Haidar et al. AAC 2017;e02534-16

Lahiri et al. AAC 2017; 61:e00079-17

- blaKPC-2 mutationsCompain et al. AAC 2017; 61:e00451-17; Barnes et al. mbio 2017; 8:e00528-17

▪ Increase hydrolitic activity of bla CTX-M-14 variantsBoth et al.J Antmicrob Chemother 2017; 72: 2483–8

Restoration of carbapenem susceptibility, expression as an ESBL

Emergence of CAZ-AVI resistance and restoration of

carbapanem susceptibility in KPC producing K. pneumoniae

Time-kill responses of CAZ-AVI

resistant Klebsiella pneumoniae to

meropenem (● no drug, ▲ 4xMIC,

x 8xMIC, ■ 16 μg/mL)

Etest results for meropenem and

CAZ-AVI against baseline isolate

(left) and follow-up isolate (right)

▪ Restoration of meropenem susceptibility among CAZ-AVI resistant

K. pneumoniae during CAZ-AVI treatment

Shields et al. Open Forum Infect Dis. 2017; 4(3):ofx101

▪ In-vitro selection of CTL-TAZ resistance requires multiple mutations

leading to over expression and structural modifications of AmpC

Cabot et al. AAC 2014; 58:3091-9

▪ In-vivo emergence of CTL-TAZ resistance in CTL-TAZ treated

patients

- in isolates resistant to carbapenems due to OprD mutation (Q142X)

and other β-lactams due to AmpR mutation (G154R) leading AmpC

over expression and acquiring

- AmpC mutations (E247K, T96I) or deletion of 19 amino

acids (G229-E247)

- in isolates with OXA-10 and emergence of OXA-14 due to new

mutations (N146S)

- in isolates with OXA-2 and emergence of OXA-539 due to D149

duplication

MacVane et al. AAC 2017; 61: e01183-17; Fraile-Ribot et al, AAC 2017; 61(9). pii: e01117-17

Fraile-Ribot et al. JAC 2018; 73:658–3

Emergence of ceftolozane-tazobactam (TZC) acquired

resistance in Pseudomonas aeruginosa

▪ In-vitro selection of TZC resistance requires multiple mutations leading

to over expression and structural modifications of AmpCCabot et al. AAC 2014; 58:3091-9

▪ In-vivo emergence of TZC resistance in TZC treated patients

MacVane et al. AAC 2017; 61: e01183-17; Fraile-Ribot et al, AAC 2017; 61(9). pii: e01117-17

Fraile-Ribot et al. JAC 2018; 73:658-3; Díaz et al. EJCMID 2018;

AmpC-high (new mutations/

deletion) + OprD low

CAZ: 16-32 mg/L

IMP: 16 mg/L

TZC: 32->32 mg/L

OXA-14 / OXA-53, OprD-low

CAZ: >64 mg/L

IMP: 16 mg/L

TZC: 32 mg/L

WT

CAZ: 2 mg/L

IMP: 1 mg/L

TZC: 0.5 mg/L

AmpC-high +

OprD-low

CAZ: 16-32 mg/L

IMP: 16 mg/L

TZC: 2 mg/L

OXA-2 / OXA-10

CAZ: 2 mg/L

IMP: 2 mg/L

TZC: 4 mg/L

Emergence of ceftolozane-tazobactam (TZC) acquired

resistance in Pseudomonas aeruginosa

▪ Why new β-lactam-β-lactamase inhibitor combinations?

▪ Which are the new β-lactam-β-lactamase inhibitor combinations?

▪ How the new β-lactam-β-lactamase inhibitor combinations work?

▪ How is the resistant development? How I can detect it?

▪ How can avoid emergence of resistance?

2018

Ceftazidime/avibactam recomendation Grading

Could use as an alternative to carbapenems for infection with ESBL and

AmpC-producing Enterobacteriaceae but alternatives may be cheaper.

Conditional

for

Evaluate further use alone or in combination when non-MBL

carbapenemase-producing organisms cause infection. KPC-3-producing

Klebsiella are vulnerable to mutations in the enzyme causing resistance.

Research

and trials

Consider whether it should be used with a carbapenem or colistin to treat

infections with KPC-3 producers based on latest evidence at the time of use.

Research

and trials

Do not use for treating infection with anaerobes or bacteria producing MBLs:

these are resistant.

Strong

against

▪ Independent emergence of resistant phenotypes

(Ph2 and Ph3) from the susceptible one (Ph1)

. MIC (mg/L) .

Day Ph CAZ-AVI MER Sample .

5 Ph1 S (3) R (128) Pancratic fluid

49 Ph2* R (>256) S (2) BAL

. 68 Ph3** R (12) R (>128) Blood .

*D179Y mutation in KPC-2;

**porin deficiency (C1049T mutation in ompK35

and IS1 insertion 49bp upstram ompK36)

▪ Treatment (days): MER 1-----------38 60-------------72

PMB 7-----36 57---------------72

TGC 24---------------------------------60

CAZ-AVI 37-----------------------------60

Ph2(49) Ph3(68)Ph1 (5)

▪ The combination did not suppress

ceftazidime-avibactam resistance

▪ Colistin + ceftazidime-avibactam

did not provide a benefit over

ceftazidime-avibactam alone

against most CRE isolates.

2018

Ceftolozane/tazobactam recomendations Grading

Use to treat susceptible infections with P. aeruginosa resistant to

ceftazidime.

Conditional for

Use as an alternative to carbapenems to treat urinary or intraabdominal

infection involving ESBL-producing E. coli. Caution may be needed when

treating infections with ESBL-producing Klebsiella spp. owing to a higher

resistance rate.

Conditional for

Do not use for infections due to AmpC- or carbapenemase producing

Enterobacteriaceae or MBL/ESBL-producing P. aeruginosa.

Strong against

Risk of resistance development is increased when

P. aeruginosa have increased ceftazidime MICs

▪ New β-lactam-β-lactamase inhibitor combinations are needed to

address current resistance landscape

▪ The use of this new combinations does not exclude the possibility

of resistance development

▪ Resistance development of new β-lactam-β-lactamase inhibitor

combinations can be phenotypically recognized

▪ Resistance development of new β-lactam-β-lactamase inhibitor

combinations can not be excluded when using in combination

with other antibiotic classes

Acknowledgements

Departamento de

Microbiología II

Universidad

Complutense. Madrid

Hospital Universitario Ramón y CajalSERVICIO DE MICROBIOLOGÍA Y PARASITOLOGÍA

Dr. Rafael Cantón

Resistance to new β-lactam-

β-lactamase inhibitor

combinations: a new step in

the muti-drug resistance?