In vitro activity of imipenem/relebactam against Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal (SUPERIOR and STEP studies)

Abstract

Introduction

The increasing rates of resistance to currently available antimicrobial agents among Gram-negative bacterial isolates is a great concern worldwide, particularly in ICUs.^1,2 In the last decade, the worldwide spread of MDR and XDR Pseudomonas aeruginosa isolates associated with significantly increased morbidity and mortality has become a public health threat and calls for the development of novel antimicrobial agents. In P. aeruginosa, resistance mechanisms to antipseudomonal β-lactam antibiotics comprise mutations in chromosomal genes and transferable resistance determinants, including carbapenemase-encoding genes. The successful dissemination of the so-called high-risk clones of MDR/XDR P. aeruginosa has also contributed to the adaptation and establishment of these pathogens in the healthcare setting, causing severe infections with compromised treatment options.^3–7

Imipenem/relebactam is a novel β-lactam/β-lactamase inhibitor combination indicated for the treatment of complicated urinary tract infections (cUTIs), complicated intra-abdominal infections (cIAIs) and hospital-acquired (HAP) and ventilator-associated bacterial pneumonia (VAP) in adult patients, with limited or no alternative therapeutic options.^8,9 Imipenem/relebactam shows activity against MDR isolates, including class A and class C β-lactamase-producing Enterobacterales and carbapenem-resistant P. aeruginosa isolates.^8,9 Furthermore, the addition of relebactam has showed a significant benefit in potentiating the activity of imipenem against imipenem-non-susceptible P. aeruginosa with OprD deficiency and AmpC overexpression.^10,11

In the present study, we evaluated the in vitro activity of imipenem/relebactam and comparator agents against a well-characterized large collection of P. aeruginosa clinical isolates prospectively recovered from ICU patients in Portugal and Spain during two surveillance studies (STEP and SUPERIOR, respectively). Using WGS, we also characterized the population structure and the resistome of a subset of P. aeruginosa isolates, focusing on the resistance mechanisms possibly affecting imipenem/relebactam activity.

Materials and methods

Background and study design

STEP and SUPERIOR surveillance studies were designed to determine the in vitro activity of ceftolozane/tazobactam, imipenem/relebactam and comparator agents against P. aeruginosa and Enterobacterales clinical isolates prospectively collected from patients admitted to 11 Portuguese (STEP, 2017–18) and 8 Spanish (SUPERIOR, 2016–17) ICUs. A total of 474 P. aeruginosa (STEP, n = 395; SUPERIOR, n = 79) non-replicate clinical isolates causing cUTIs [STEP (22.8%; 90/395); SUPERIOR (58.2%; 46/79)], cIAIs [STEP (20.2%; 80/395); SUPERIOR (41.8%; 33/79)] or LRTIs [STEP (56.9%; 225/395)] were recovered.^12,13

A subset of 62 P. aeruginosa isolates (STEP, n = 48; SUPERIOR, n = 14) were selected for the subsequent genome analysis using WGS. All P. aeruginosa isolates that showed imipenem/relebactam-resistant MIC values (n = 30) based on the EUCAST 2022 interpretative criteria were selected (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_12.0_Breakpoint_Tables.pdf). For a proper comparison, another 32 imipenem/relebactam-susceptible P. aeruginosa isolates, previously characterized during the STEP and SUPERIOR studies, were also included in the genome analysis.¹⁴

Antimicrobial susceptibility testing and resistance phenotypes

Antimicrobial susceptibility testing was performed at the central laboratory by the reference broth microdilution method (standard ISO method 20776-1:2006). The antimicrobials and range of concentrations tested were as follows: ceftazidime/avibactam (0.03/4–64/4 mg/L), ceftolozane/tazobactam (0.03/4–32/4 mg/L), imipenem/relebactam (0.03/4–64/4 mg/L) and imipenem (0.03–64 mg/L). Quality control testing was performed using the P. aeruginosa ATCC 27853 strain. MIC values were interpreted in accordance with the EUCAST 2022 (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_12.0_Breakpoint_Tables.pdf) and CLSI 2020 (https://clsi.org/standards/products/microbiology/documents/m100/) guidelines. The EUCAST 2022 clinical breakpoint used for imipenem/relebactam was: susceptible, MIC_IPM ≤ 2/4 mg/L; and resistant, MIC_IPM> 2/4 mg/L. The CLSI clinical breakpoints for imipenem/relebactam were: susceptible, MIC ≤ 2/4 mg/L; intermediate, MIC = 4/4 mg/L; and resistant, MIC ≥ 8/4 mg/L. Based on our previous antibiotic susceptibility studies, the following resistant phenotypes were defined: TZP/CAZ-R (combined piperacillin/tazobactam and ceftazidime resistance) and TZP/CAZ/MEM-R (combined piperacillin/tazobactam, ceftazidime and meropenem resistance).^12,13 Antimicrobial susceptibility patterns of P. aeruginosa were characterized as per recommended guidelines: MDR (non-susceptible to at least one agent in three or more antimicrobial categories); XDR (non-susceptible to at least one agent in all but two or fewer antimicrobial categories, i.e. bacterial isolates that remained susceptible to only one or two categories); pandrug-resistant (PDR, non-susceptible to all agents in all antimicrobial categories); and non-MDR.¹⁵ The difficult-to-treat resistance (DTR) phenotype was also applied to P. aeruginosa isolates that were intermediate or resistant to all β-lactam categories, including carbapenems, and fluoroquinolones.¹⁶

WGS and bioinformatics analysis

Genomic DNA extraction was carried out using the commercial chemagic DNA Bacterial External Lysis Kit (PerkinElmer, USA). WGS was performed using the Illumina NovaSeq 6000 platform (Oxford Genomics Centre, Oxford, UK), with 2 × 150 bp paired-end reads. Sequencing processing, molecular typing, screening for acquired resistome and analysis of chromosomal determinants involved in antibiotic resistance were carried out as described previously.¹⁴ All complete sequences were deposited at DDBJ/ENA/GenBank under BioProject accession number PRJNA629475 (Table S1, available as Supplementary data at JAC Online).

Ethics

The STEP study was approved by the Ethical Committee of all participating Portuguese hospitals. The SUPERIOR study was approved by the Ethical Committee of Hospital Universitario Ramón y Cajal (Madrid, Spain) (Ref. 087-16) and the Spanish Medicines Agency (Ref. MSD-CEF-2016-01).

Results

Antimicrobial susceptibility

Overall, imipenem/relebactam and comparators showed comparable activity: imipenem/relebactam (93.7% susceptible by EUCAST and CLSI); ceftazidime/avibactam (93.5% susceptible by EUCAST and CLSI); and ceftolozane/tazobactam (93.2% susceptible by EUCAST and CLSI). Imipenem activity did not exceed 72% by EUCAST [71.9% categorized as susceptible, increased exposure (I)] and 66% by CLSI [65.8% categorized as susceptible]. Using MIC₉₀ values, imipenem/relebactam (MIC₉₀= 2 mg/L) was 2-fold more active than ceftolozane/tazobactam (MIC₉₀= 4 mg/L), 4-fold more active than ceftazidime/avibactam (MIC₉₀= 8 mg/L) and 8-fold more active than imipenem (MIC₉₀= 16 mg/L) (Table 1). Overall, up to 21.5% of the isolates were classified as MDR, 23.8% as XDR and 18.6% as DTR (Table 1).

The imipenem/relebactam resistance rate was 6.3% (30/474) by EUCAST and 5.1% (24/474) by CLSI criteria (Table 1). Differences were found between countries by both EUCAST (Portugal, 5.8% resistant; Spain, 8.9% resistant) and CLSI (Portugal, 4.8% resistant; Spain, 6.3% resistant) criteria (Table 1). By infection source, imipenem/relebactam showed high susceptibility rates (89.7% susceptible in cUTIs, 90.3% susceptible in cIAIs and 97.8% susceptible in LRTIs), although it was especially active in those isolates recovered from LRTIs, regardless of the resistance phenotype (88.9%–100% susceptible) (Table 2).

As shown in Table 1, susceptibility to imipenem/relebactam of isolates with resistant phenotypes ranged from 72.3% to 94.1%. Using MIC₅₀ and MIC₉₀ values, imipenem/relebactam was more active than ceftazidime/avibactam and ceftolozane/tazobactam in all subsets of resistance phenotypes (Table 1).

On the other hand, among the P. aeruginosa strains with resistant MIC values for ceftazidime/avibactam (31/474), ceftolozane/tazobactam (32/474) or both ceftazidime/avibactam plus ceftolozane/tazobactam (14/474), the rate of imipenem/relebactam-susceptible isolates was 64.5% (20/31), 28.1% (9/32) and 35.7% (5/14), respectively.

In addition, relebactam restored the activity of imipenem in 76.9% (103/134) of P. aeruginosa isolates with imipenem-resistant MIC values, including MDR (82.1%; 32/39), XDR (68.8%; 53/77) and DTR (67.2%; 45/67) isolates (Figure 1).

Figure 1.

Distribution of P. aeruginosa isolates recovered during the STEP and SUPERIOR surveillance studies by resistance phenotype (MDR/XDR and DTR) and the imipenem/relebactam and imipenem MIC values. Dotted lines represent the EUCAST 2022 clinical breakpoints of imipenem/relebactam (susceptible, MIC ≤ 2/4 mg/L; resistant, MIC > 2/4 mg/L) and imipenem (susceptible, MIC ≤ 0.001 mg/L; resistant, MIC > 4 mg/L). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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Population structure and acquired resistance genes

Among the sequenced P. aeruginosa strains (STEP, n = 48; SUPERIOR, n = 14), differences in population structure were detected depending on the country of origin. In Portugal, the most frequent clone was the clonal complex CC235 (n = 15), followed by CC244 (n = 7), CC348 (n = 5), CC253 (n = 3) and CC179 (n = 3). In Spain, CC175 was predominant (n = 11). Different carbapenemase gene distributions were also found: GES-13 (n = 13), VIM-2 (n = 3) and KPC-3 (n = 2) in Portugal; and VIM-20 (n = 3), VIM-1 (n = 2), VIM-2 (n = 1) and VIM-36 (n = 1) in Spain. GES-13-CC235 (n = 13) and VIM type-CC175 (n = 5) associations were predominant in Portugal and Spain, respectively (Figure 2). It is noteworthy that all GES-13-CC235 isolates were associated with XDR and DTR profiles. Among other frequent clones, the XDR/DTR profile was also found (5/11 CC175, 4/7 CC244, 5/5 CC348, 2/3 CC179) (Figure 2). The presence of other horizontally acquired β-lactamase genes was confirmed, such as bla_OXA-1 (4/62) in non-carbapenemase producers and bla_OXA-2 (4/62) in VIM-20-producing P. aeruginosa strains (3/4) (Table S2). Overall, a clustering of clones by the horizontal acquired gene content was observed (Table S2).

Figure 2.

Similarity tree of P. aeruginosa isolates sequenced (n = 62) during the STEP and SUPERIOR surveillance studies with epidemiological and molecular data (CC and carbapenemase gene content) obtained by WGS. Resistance phenotype classification (MDR/XDR and DTR) and imipenem/relebactam (IMR) [susceptible (S), MIC ≤ 2/4 mg/L; resistant (R), MIC > 2/4 mg/L] and imipenem (IPM) (S, MIC ≤ 0.001 mg/L; R, MIC > 4 mg/L) susceptibility results interpreted according to EUCAST 2022 criteria are also included. Branch length is indicative of the MASH distance. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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Overall, among carbapenemase producers (40.3%; 25/62), 84% (21/25) of the isolates showed an imipenem/relebactam-resistance phenotype (MIC_IMR= 8 to >64 mg/L). All KPC-3 P. aeruginosa strains were susceptible to imipenem/relebactam (2/2); but all GES-13-CC235 (13/13) (MIC_IPM= 8 mg/L) and most of the VIM producers (8/10) [4/4 VIM-2 (2 CC244, 1 CC175, 1 CC179), 3/3 VIM-20 (3 CC175), 1/2 VIM-1 (1 CC309)] (MIC_IPM= 32 to >64 mg/L) were categorized as resistant (Figure 2). Among non-carbapenemase-producing strains (59.7%; 37/62), imipenem/relebactam susceptibility was 75.7% (28/37). Nine non-carbapenemase-producing P. aeruginosa isolates (24.3%; 9/37) belonging to different clones (2 CC175, 1 CC179, 1 CC554, 1 CC1239, 1 CC2123, 1 CC395, 1 CC606 and 1 CC884) were resistant to imipenem/relebactam (MIC_IPM= 4–16 mg/L). Other additional genes involved in resistance to fluoroquinolones and aminoglycosides were found in these isolates (8/9 crpP, 8/9 aph3-IIb, 2/9 aadB and 1/9 aac6-Ib-cr) (Table S2).

Mutational resistome

Results of all mutations (non-synonymous SNPs and insertions/deletions) detected in chromosomal genes known to be related to antimicrobial resistance in P. aeruginosa isolates are included in Table S2. As shown in Figure S1, specific mutational resistome patterns were observed for the different P. aeruginosa clones when analysing a subset of genes suspected to be involved in the resistance to imipenem/relebactam. Among these selected genes, the most frequently mutated targets included: Opr porin genes (oprD, 90%); QRDR genes (gyrA, 74%; parC, 55%); AmpC regulators (ampD, 44%); efflux pump-encoding genes and regulators (mexB, 29%; mexR, 16%; mexS, 16%; mexZ, 52%; armZ, 15%); and PBP genes (ftsI, 15%). In the subset of P. aeruginosa strains with an imipenem/relebactam-resistance profile, the most frequent mutated genes were: Opr porin genes (oprD, 90%; oprN, 13%); QRDR genes (gyrA, 80%; parC, 70%); AmpC regulators (ampD, 47%); efflux pump-encoding genes and regulators (mexB, 50%; mexR, 13%; mexZ, 73%; armZ, 13%); and LPS modification genes (parS, 13%) (Figure S1). In addition, specific mutations were found in these genes that only affected imipenem/relebactam-resistant P. aeruginosa isolates in which carbapenemase genes were not detected (Table 3). These mutations were mainly encountered in efflux pump-encoding genes and regulators (6/9; mexB, mexR, mexS, mexT, mexY and nalC), LPS modification genes (5/9; parS, pmrA and pmrB), Opr genes (4/9; oprD and oprN), QRDR genes (1/9; parC), AmpC regulators (1/9; dacB and mpl) and PBP genes (1/9; ftsI) (Table 3). Interestingly, two isolates showed the mexR T130P substitution, previously related to imipenem/relebactam resistance (Table 3).¹⁷

Discussion

P. aeruginosa is one of the most frequent Gram-negative opportunistic pathogens causing severe respiratory infections, particularly in ICUs and immunocompromised patients. P. aeruginosa is responsible for 10% of all nosocomial infections and is also increasing as a cause of community-acquired infections. Furthermore, the high rates of antimicrobial resistance reported worldwide in clinical strains of P. aeruginosa that cause severe infections with very few treatment options led this species to be considered an ESKAPE pathogen.¹⁸ In addition, carbapenem-resistant P. aeruginosa isolates are included in the WHO priority list for which research and development of new antibiotics is critical.¹⁹

During the STEP and SUPERIOR studies, imipenem/relebactam showed excellent activity against P. aeruginosa clinical isolates (94%), comparable with that found between 2015 and 2017 in the USA (94%) and in Europe (92%) as a part of the SMART surveillance programme.^20,21 In 2019, Fraile-Ribot et al.²² reported a similar imipenem/relebactam susceptibility rate (97%) against a large collection of P. aeruginosa isolates recovered in 2017 from 51 Spanish hospitals. Imipenem/relebactam has also been demonstrated to be a useful alternative in the treatment of MDR/XDR P. aeruginosa infections, also potentially reducing resistance development during the antibiotic therapy.^22,23 In our collection, imipenem/relebactam was highly active against the subsets of MDR (94%), XDR (79%) and DTR P. aeruginosa isolates (75%). It should be noted that these figures are higher than those found in MDR P. aeruginosa isolates (78% susceptible) collected in Europe as part of the SMART study.²¹ Furthermore, in our study, in contrast to the SMART study, no differences by type of infection were found; imipenem/relebactam susceptibility was particularly high in the P. aeruginosa isolates recovered from LRTI, regardless of the resistance phenotype.²¹

On the other hand, previous studies demonstrated that imipenem/relebactam displays good activity against imipenem-resistant P. aeruginosa isolates with deficient OprD and increased AmpC expression.^10,11 Moreover, relebactam has been demonstrated to be effective in restoring the imipenem activity against carbapenem-resistant P. aeruginosa strains by inhibiting the AmpC β-lactamase.⁹ In our collection, coinciding with previous data, relebactam restored the activity of imipenem in nearly 80% of imipenem-resistant P. aeruginosa isolates.^20–22 Additionally, Fraile-Ribot et al.²² have also demonstrated that imipenem/relebactam exhibits good activity against P. aeruginosa isolates that have developed resistance to ceftazidime/avibactam and ceftolozane/tazobactam during the antibiotic treatment. Interestingly, during the STEP and SUPERIOR studies, imipenem/relebactam showed a high rate of activity against the subset of ceftazidime/avibactam-resistant P. aeruginosa isolates (65%).

The success of P. aeruginosa as a major pathogen in the hospital setting is mainly due to the versatility and adaptive character of its genome, distinguished by its ability to select chromosomal genetic mutations, but also to rapidly develop acquired resistance as a consequence of antibiotic exposure.^24,25 Furthermore, MDR/XDR P. aeruginosa isolates that are responsible for epidemic outbreaks and increased morbidity and mortality rates usually belong to prevalent and well-disseminated high-risk clones.^4,5 In the last decade, ST235 and ST175 high-risk clones of P. aeruginosa have emerged globally as major contributors of hospital-acquired infections, due to their ability to acquire and maintain antibiotic resistance elements, particularly those encoding carbapenemases.^6,26–28 In our study, among the sequenced strains, both GES-13-CC235 and VIM type-CC175 high-risk clones were the most frequent P. aeruginosa isolates in Portugal and Spain, respectively, and were mainly related to an imipenem/relebactam-resistance profile. Moreover, coinciding with previous data, an elevated content of genes involved in resistance to aminoglycosides was also detected in both CC235 and CC175 clones.^26,28 Furthermore, the GES-13-CC235 high-risk clone was highly associated with the XDR/DTR-resistance profile. Along with CC235 and CC175, we found other MDR/XDR P. aeruginosa clones such as CC244, CC253, CC348, CC309 and CC179, frequently detected in Europe, including Portugal and Spain.^29–32 Overall, resistance to imipenem/relebactam in these clones was mainly associated with VIM-type carbapenemase production.

According to our results, imipenem/relebactam resistance in P. aeruginosa is mainly due to the presence of carbapenemase genes. However, mutations leading to the inactivation of porins, overexpression of efflux pumps and LPS modification, among others, could be involved also. In fact, among the imipenem/relebactam-resistant non-carbapenemase-producing P. aeruginosa isolates, specific mutations were found in genes previously related to an imipenem/relebactam-resistance profile.^17,23 It is also remarkable that these mutations were mainly found in XDR/DTR P. aeruginosa isolates that did not belong to high-risk clones. Interestingly, our genome analysis revealed that two of these isolates showed the mutation T130P in the efflux pump regulator gene mexR, also previously reported as a potential mechanism for resistance to imipenem/relebactam in the ST175 high-risk clone.¹⁷

In conclusion, imipenem/relebactam showed excellent overall activity against MDR/XDR P. aeruginosa clinical isolates causing complicated infections in ICU patients from Portugal and Spain. Imipenem/relebactam activity was maintained regardless of the resistance phenotype, including MDR, XDR, DTR isolates and the subset of ceftazidime/avibactam-resistant P. aeruginosa isolates. Imipenem/relebactam resistance was mainly associated with GES-13 and VIM-type carbapenemase production; however, coincident mutations affecting the inactivation of porins, overexpression of efflux pump and LPS modification may also be involved and need to be further studied.

Acknowledgements

Members of the STEP study group

José Melo-Cristino (Centro Hospitalar Lisboa Norte, Lisboa, Portugal); Margarida F. Pinto, Cristina Marcelo, Helena Peres, Isabel Lourenço, Isabel Peres, João Marques, Odete Chantre and Teresa Pina (Centro Hospitalar Universitário Lisboa Central, Lisboa, Portugal); Elsa Gonçalves and Cristina Toscano (Centro Hospitalar de Lisboa Ocidental, Lisboa, Portugal); Valquíria Alves (Unidade Local de Saúde de Matosinhos, Matosinhos, Portugal); Manuela Ribeiro, Eliana Costa and Ana Raquel Vieira (Centro Hospitalar Universitário São João, Porto, Portugal); Sónia Ferreira, Raquel Diaz and Elmano Ramalheira (Hospital Infante Dom Pedro, Aveiro, Portugal); Sandra Schäfer, Luísa Tancredo and Luísa Sancho (Hospital Prof. Dr. Fernando Fonseca, Amadora, Portugal); Ana Rodrigues and José Diogo (Hospital Garcia de Orta, Almada, Portugal); Rui Ferreira (Unidade de Portimão, Portugal); Hugo Cruz, Helena Ramos, Tânia Silva and Daniela Silva (Centro Hospitalar Universitário do Porto, Porto, Portugal); Catarina Chaves, Carolina Queiroz and Altair Nabiev (Centro Hospitalar Universitário de Coimbra, Coimbra, Portugal); and Joana Duarte, Leonor Pássaro and Carolina Moura (MSD Portugal, Paço de Arcos, Portugal).

Members of the SUPERIOR study group

Antonio Oliver and Xavier Mulet (Hospital Universitario Son Espases, Palma de Mallorca, Spain); Emilia Cercenado (Hospital General Universitario Gregorio Marañón, Madrid, Spain); Germán Bou and M. Carmen Fernández (Hospital Universitario A Coruña, A Coruña, Spain); Álvaro Pascual and Mercedes Delgado (Hospital Universitario Virgen Macarena, Sevilla, Spain); Concepción Gimeno and Nuria Tormo (Consorcio Hospital General Universitario de Valencia, Valencia, Spain); Jorge Calvo, Jesús Rodríguez-Lozano and Ana Ávila Alonso (Hospital Universitario Marqués de Valdecilla, Santander, Spain); Jordi Vila, Francesc Marco and Cristina Pitart (Hospital Clínic, Barcelona, Spain); María García del Castillo, Sergio García-Fernández, Marta Hernández-García and Rafael Cantón (Hospital Universitario Ramón y Cajal, Madrid, Spain); and Jazmín Díaz-Regañón (MSD España, Madrid, Spain).

Funding

The study was funded by MSD Portugal (protocol VP6918) and MSD Spain (protocol MSD-CEF-2016-01). This study was also supported by Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (RD16/0016/0001, RD16/0016/0004, RD16/0016/0006, RD16/0016/0007, RD16/0016/0010 and REIPI RD16/0016/0011), co-financed by the European Development Regional Fund ‘A way to achieve Europe’ (ERDF), Operative program Intelligent Growth 2014–2020 and CIBER de Enfermedades Infecciosas (CIBERINFEC) (CB21/13/00084), Instituto de Salud Carlos III, Madrid, Spain.

Transparency declarations

Rafael Canton and Antonio Oliver have participated in educational programmes and research grants organized by MSD, Pfizer and Shionogi. José Melo-Cristino, German Bou and Emilia Cercenado have participated in educational programmes organized by MSD and Pfizer. Margarida F. Pinto had a travel grant for ECCMID 2019 from MSD Portugal. Joana Duarte is an MSD Portugal employee and/or may hold stock options in Merck & Co., Inc., Kenilworth, NJ, USA. Jazmín Díaz-Regañón is an employee of MSD Spain. All other authors declare no competing interests.

Supplementary data

Figure S1 and Tables S1 and S2 are available as Supplementary data at JAC Online.

References

Author notes