Abstract
Objectives
To investigate the in vitro activity of ceftazidime/avibactam and ceftolozane/tazobactam against clinical isolates of MDR Pseudomonas aeruginosa from Qatar, as well as the mechanisms of resistance.
Methods
MDR P. aeruginosa isolated between October 2014 and September 2015 from all public hospitals in Qatar were included. The BD PhoenixTM system was used for identification and initial antimicrobial susceptibility testing, while Liofilchem MIC Test Strips (Liofilchem, Roseto degli Abruzzi, Italy) were used for confirmation of ceftazidime/avibactam and ceftolozane/tazobactam susceptibility. Ten ceftazidime/avibactam- and/or ceftolozane/tazobactam-resistant isolates were randomly selected for WGS.
Results
A total of 205 MDR P. aeruginosa isolates were included. Of these, 141 (68.8%) were susceptible to ceftazidime/avibactam, 129 (62.9%) were susceptible to ceftolozane/tazobactam, 121 (59.0%) were susceptible to both and 56 (27.3%) were susceptible to neither. Twenty (9.8%) isolates were susceptible to ceftazidime/avibactam but not to ceftolozane/tazobactam and only 8 (3.9%) were susceptible to ceftolozane/tazobactam but not to ceftazidime/avibactam. Less than 50% of XDR isolates were susceptible to ceftazidime/avibactam or ceftolozane/tazobactam. The 10 sequenced isolates belonged to six different STs and all produced AmpC and OXA enzymes; 5 (50%) produced ESBL and 4 (40%) produced VIM enzymes.
Conclusions
MDR P. aeruginosa susceptibility rates to ceftazidime/avibactam and ceftolozane/tazobactam were higher than those to all existing antipseudomonal agents, except colistin, but were less than 50% in extremely resistant isolates. Non-susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam was largely due to the production of ESBL and VIM enzymes. Ceftazidime/avibactam and ceftolozane/tazobactam are possible options for some patients with MDR P. aeruginosa in Qatar.
Introduction
Pseudomonas aeruginosa remains a leading cause of hospital-acquired infections including those of the bloodstream, respiratory tract, urinary tract and surgical sites.1–3 In addition to an array of virulence determinants, P. aeruginosa possesses and can readily acquire a broad variety of antimicrobial resistance mechanisms.4,5 These include up-regulation of efflux pumps, loss of outer membrane porins, the production of AmpC, ESBL and carbapenemase enzymes, and modification of antimicrobial target sites.6,7 Multiple resistance mechanisms are usually expressed simultaneously, resulting in resistance to agents in multiple antimicrobial classes.6,7 The existence of limited effective treatment options for MDR P. aeruginosa infections has been associated with poor clinical outcomes.8–10 In their 2017 report, the WHO designated research, discovery and development of new antibiotics for carbapenem-resistant P. aeruginosa a critical priority.11
Ceftazidime/avibactam and ceftolozane/tazobactam are licensed for the treatment of patients with a variety of clinical infections.12 Avibactam is a non-β-lactam β-lactamase inhibitor that inhibits class A, class C and most class D β-lactamases.13 On the other hand, ceftolozane is a novel cephalosporin that is active against P. aeruginosa isolates with AmpC hyperproduction and overexpressed efflux mechanisms.14 The combination of ceftolozane and the β-lactamase inhibitor tazobactam is active against many, but not all, ESBL-producing Gram-negative bacteria.15 Several studies have reported rates and mechanisms of P. aeruginosa resistance to ceftazidime/avibactam and ceftolozane/tazobactam, including MDR isolates, from Europe and North America.15–20 However, there are limited data on the potential utility of ceftazidime/avibactam and ceftolozane/tazobactam for MDR P. aeruginosa from the Arabian Peninsula, a region of extremely diverse demography and close travel links to all corners of the world.21,22 The aim of this study was to investigate the in vitro activity of ceftazidime/avibactam and ceftolozane/tazobactam against MDR P. aeruginosa from Qatar and to explore the associated genetic diversity and mechanisms of resistance.
Methods
Ethics
This study was approved with a waiver for informed consent by the Institutional Review Board, Hamad Medical Corporation, Doha, Qatar (IRGC-01-51-033) and Swedish Research Council Formas (Dn. 219-2014-837).
Materials and setting
This prospective evaluation was conducted on routine clinical specimens received by the Microbiology Laboratory at Hamad Medical Corporation, Doha during the period from 1 October 2014 to 30 September 2015, prior to any clinical use of ceftazidime/avibactam or ceftolozane/tazobactam in Qatar. The facility provides routine and tertiary diagnostic services to all public acute and referral hospitals across Qatar.
The isolates underwent standard diagnostic work-up then were stored at −70°C pending further analysis. MDR P. aeruginosa isolates were defined as having in vitro resistance to at least one agent from three or more antimicrobial classes.23
Identification and susceptibility testing
The BD PhoenixTM automated system was used for bacterial identification and initial antimicrobial susceptibility testing, while Liofilchem® MIC Test Strips (Liofilchem, Roseto degli Abruzzi, Italy) were used for confirmation of ceftazidime/avibactam and ceftolozane/tazobactam susceptibility. Escherichia coli ATCC 25922, E. coli ATCC 35218 and P. aeruginosa ATCC 27853 were used as controls. Susceptibility reporting was based on current recommendations of the CLSI.24 No intermediate susceptibility category was available for ceftazidime/avibactam against P. aeruginosa. Isolates were therefore described as susceptible to ceftazidime/avibactam if the MIC was ≤8 mg/L and non-susceptible if the MIC was >8 mg/L.24 For consistency, intermediate and resistant categories were grouped together as non-susceptible for all reported antimicrobial agents.
WGS
Ten MDR P. aeruginosa isolates that were resistant to ceftazidime/avibactam and/or ceftolozane/tazobactam were randomly selected to undergo WGS using the Illumina HiSeq 2000 system (Illumina, San Diego, CA, USA). WGS was performed by Eurofins GATC Biotech GmbH, Konstanz, Germany.
Genomic assembly, annotation and identification
The clean reads were assembled using SPAdes, Version 3.13.0 (Center for Algorithmic Biotechnology, St Petersburg, Russia). To determine whether the GC content had a significant effect on sequencing randomness or not, the GC content and average depth of the genomic sequence were calculated without repetition as a unit of 500 bp.25 The assembled data were subjected to RAST annotation, as previously described.26 MDR P. aeruginosa isolates were subjected to SpeciesFinder 1.2 (Center for Genomic Epidemiology, Lyngby, Denmark) to determine their 16S rRNA-based species identification.27
In silico serotyping
In silico serotyping of the P. aeruginosa isolates was performed using P. aeruginosa serotyper (PAst) Version 1.0 (Center for Genomic Epidemiology, Lyngby, Denmark). The programme utilizes sequencing data and is based on Basic Local Alignment Search Tool (BLAST) analysis of the OSA gene.28
MLST
MLST 1.8 (Center for Genomic Epidemiology) was used to perform MLST of MDR P. aeruginosa isolates, based on the seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA and trpE), as previously described.29
Antibiotic resistance genes
Antibiotic resistance genes were predicted using the Comprehensive Antibiotic Resistance Database (CARD), Version 1.2.0 (McMaster University, Hamilton, Ontario).30
Statistical analysis
Susceptibility patterns of MDR P. aeruginosa to the study antibiotics were presented as frequency and percentages. Cohen’s Kappa (k) was used to measure agreement between ceftazidime/avibactam and ceftolozane/tazobactam susceptibility results and those of other agents. Type I error threshold of 0.05 was used for statistical significance. Statistical analyses were conducted using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp, Armonk, NY, USA).
Results
P. aeruginosa was isolated from a total of 2533 clinical samples over the study period, of which 205 (8.1%) fulfilled the MDR definition. Respiratory cultures (92, 44.9%) were the most common source of MDR P. aeruginosa isolates, followed by skin and soft tissue (54, 26.3%), urine (48, 23.4%), blood (5, 2.4%), sterile body fluids (4, 2.0%) and vascular line tips (2, 1.0%).
Antimicrobial susceptibility patterns of MDR P. aeruginosa isolates
Antimicrobial susceptibility results and MIC distributions for 205 MDR P. aeruginosa isolates are summarized in Table 1. One hundred and forty-one (68.8%) of the isolates were susceptible to ceftazidime/avibactam, 129 (62.9%) were susceptible to ceftolozane/tazobactam, 121 (59.0%) were susceptible to both and 56 (27.3%) were susceptible to neither agent. Twenty (9.8%) isolates were susceptible to ceftazidime/avibactam but not to ceftolozane/tazobactam, and only 8 (3.9%) were susceptible to ceftolozane/tazobactam but not to ceftazidime/avibactam. Rates of susceptibility to ceftazidime/avibactam or ceftolozane/tazobactam in the presence of resistance to other antipseudomonal antibiotics is shown in Table 2. There was agreement in susceptibility results between ceftazidime/avibactam and tobramycin (k = 0.25, P < 0.001), ceftazidime/avibactam and amikacin (k = 0.27, P < 0.001), ceftolozane/tazobactam and tobramycin (k = 0.4, P < 0.001) and ceftolozane/tazobactam and amikacin (k = 0.37, P < 0.001).
Table 1.
Cumulative MIC distributions of ceftazidime/avibactam, ceftolozane/tazobactam and comparator agents for clinical MDR P. aeruginosa isolates from Qatar
| Antibiotic | Number (cumulative %) of isolates inhibited at an MIC (mg/L) of:a |
MIC50 (mg/L) | MIC90 (mg/L) | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.75 | 1 | 1.5 | 2 | 3 | 4 | 6 | 8 | 12 | 16 | 24 | 32 | 48 | 64 | 96 | 128 | 192 | 256 | |||
| CZA | 9 (4.4) | 6 (7.3) | 11 (12.7) | 24 (24.4) | 27 (37.6) | 29 (51.7) | 24 (63.4) | 11 (68.8) | 10 (73.7) | 8 (77.6) | 7 (81) | 6 (83.9) | 7 (87.3) | 4 (89.3) | 4 (91.2) | 3 (92.7) | 0 (92.7) | 15 (100) | 4 | 64 |
| C/T | 40 (19.5) | 39 (38.5) | 18 (47.3) | 13 (53.7) | 7 (57.1) | 12 (62.9) | 5 (65.4) | 3 (66.8) | 3 (68.3) | 5 (70.7) | 5 (73.2) | 2 (74.1) | 1 (74.6) | 2 (75.6) | 0 (75.6) | 0 (75.6) | 0 (75.6) | 50 (100) | 2 | 256 |
| FEP | 0 (0) | 0 (0) | 1 (0.5) | 1 (1) | 0 (1) | 1 (1.5) | 0 (1.5) | 4 (3.4) | 13 (9.8) | 16 (17.6) | 19 (26.8) | 18 (35.6) | 17 (44) | 13 (50.2) | 8 (54.1) | 8 (58) | 8 (62) | 78 (100) | 64 | 256 |
| GEN | 7 (3.4) | 7 (6.8) | 8 (10.7) | 10 (15.6) | 13 (22) | 9 (26.3) | 12 (32.2) | 17 (40.5) | 1 (41) | 6 (44) | 6 (46.8) | 4 (48.8) | 5 (51.2) | 2 (52.2) | 0 (52.2) | 0 (52.2) | 3 (53.7) | 95 (100) | 48 | 256 |
| TZP | 0 (0) | 2 (1) | 0 (1) | 1 (1.5) | 0 (1.5) | 1 (2) | 3 (3.4) | 3 (4.9) | 3 (6.3) | 7 (9.8) | 10 (14.6) | 12 (20.4) | 12 (26.3) | 12 (32.2) | 9 (36.6) | 4 (38.5) | 8 (42.4) | 118 (100) | 256 | 256 |
| AMK | 1 (0.5) | 1 (1) | 2 (2) | 2 (2.9) | 8 (6.8) | 12 (12.7) | 8 (16.6) | 19 (25.9) | 15 (33.2) | 16 (41) | 13 (47.3) | 8 (51.2) | 9 (55.6) | 6 (58.5) | 8 (62.4) | 8 (66.3) | 2 (67.3) | 67 (100) | 32 | 256 |
| TOB | 13 (6.3) | 20 (16.1) | 27 (29.3) | 18 (38) | 7 (41.4) | 7 (44.9) | 2 (45.9) | 0 (45.9) | 4 (47.8) | 1 (48.3) | 12 (54.1) | 13 (60.5) | 9 (64.9) | 5 (67.3) | 4 (69.3) | 5 (71.7) | 1 (72.2) | 57 (100) | 24 | 256 |
| CST | 43 (21) | 38 (39) | 82 (79.5) | 35 (96.6) | 3 (98) | 2 (99) | 1 (99.5) | 1 (100) | 0 (100) | 0 (100) | 0 (100) | 0 (100) | 0 (100) | 0 (100) | 0 (100) | 0 (100) | 0 (100) | 0 (100) | 1.5 | 2 |
| MEM | 8 (3.9) | 1 (4.4) | 5 (6.8) | 6 (9.8) | 4 (11.7) | 4 (13.7) | 4 (15.6) | 9 (20) | 4 (22) | 3 (23.4) | 1 (23.9) | 156 (100) | not applicable | 32 | 32 | |||||
| CIP | 12 (5.9) | 5 (8.3) | 5 (10.7) | 7 (14.1) | 9 (18.5) | 14 (25.4) | 6 (28.3) | 6 (31.2) | 6 (34.1) | 2 (35.1) | 3 (36.6) | 130 (100) | not applicable | 32 | 32 | |||||
AMK, amikacin; CIP, ciprofloxacin; CST, colistin; C/T, ceftolozane/tazobactam; CZA, ceftazidime/avibactam; FEP, cefepime; GEN, gentamicin; MEM, meropenem; TOB, tobramycin; TZP, piperacillin/tazobactam.
White, susceptible; grey, non-susceptible.
Table 2.
MDR P. aeruginosa susceptibility to ceftazidime/avibactam or ceftolozane/tazobactam in the presence of resistance to other antipseudomonal antimicrobial agents
| MDR resistance phenotype that included resistance to: | Isolates with resistance, n (%) | Isolates susceptible to CZA, n (%) | Isolates susceptible to C/T, n (%) |
|---|---|---|---|
| FEP | 198 (96.6) | 134 (67.7) | 122 (61.6) |
| TZP | 186 (90.7) | 126 (67.7) | 119 (63.9) |
| MEM | 185 (90.2) | 126 (68.1) | 140 (75.7) |
| CIP | 187 (91.2) | 142 (75.9) | 131 (70.1) |
| AMK | 119 (58.0) | 67 (56.3) | 55 (46.2) |
| GEN | 150 (73.2) | 98 (65.3) | 82 (54.7) |
| CAZ, FEP, TZP and MEM | 165 (80.5) | 106 (64.2) | 103 (62.4) |
| CAZ, FEP, TZP, MEM and CIP | 150 (73.2) | 91 (60.7) | 91 (60.7) |
| CAZ, FEP, TZP, MEM, CIP, GEN and AMK | 86 (42.0) | 38 (44.2) | 36 (41.9) |
AMK, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; C/T, ceftolozane/tazobactam; CZA, ceftazidime/avibactam; FEP, cefepime; GEN, gentamicin; MEM, meropenem; TZP, piperacillin/tazobactam.
Genotypic profile of selected MDR P. aeruginosa isolates that were non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam or both
The 10 randomly selected isolates of MDR P. aeruginosa that were non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam or both belonged to six different STs (Table 3). Class A ESBLs were identified in five (50%) isolates and Verona integron-encoded MBL (VIM) in four (40%). Genes encoding different types of Pseudomonas-derived cephalosporinases (PDCs) and oxacillinases (OXAs) were present in all of the isolates. Each isolate possessed genes for three or four different β-lactamases from three different molecular classes (Table 3). No mutations were detected in genes encoding for efflux pump regulators or efflux pump complexes in any of the 10 isolates. No shared distinctive genotypic pattern was apparent for the three isolates that were susceptible to ceftazidime/avibactam but not to ceftolozane/tazobactam (Table 3). Furthermore, none of the previously described ceftolozane/tazobactam and ceftazidime/avibactam resistance-associated PDC mutations was identified in any of the isolates.5
Table 3.
Genotypic and phenotypic profiles of 10 MDR P. aeruginosa isolates that were found to be non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam or both
| Sample number |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| PA9 | PA37 | PA99 | PA11 | PA12 | PA98 | PA123 | PA125 | PA199 | PA154 | |
| ST (serogroup) | 235 (O11) | 235 (O11) | 235 (O11) | 308 (O11) | 308 (O11) | 308 (O11) | 292 (O12) | 823 (O11) | 233 (O6) | 27 (O1) |
| β-Lactamase gene (molecular class), gene presence (% identitiy) | ||||||||||
| TEM-116 (class A) | − | − | − | − | − | − | − | − | − | yes (100) |
| VEB-1a (class A) | − | − | yes (100) | yes (100) | yes (100) | yes (100) | − | − | − | − |
| CARB-3 (class A) | − | − | − | − | − | − | yes (99.67) | − | − | − |
| VIM-2 (class B) | yes (100) | yes (100) | − | − | − | − | − | yes (100) | yes (100) | − |
| PDC-2 (class C) | yes (99.75) | yes (99.75) | yes (99.75) | − | − | − | − | − | − | − |
| PDC-3 (class C) | − | − | − | − | − | − | − | − | yes (100) | − |
| PDC-5 (class C) | − | − | − | − | − | − | yes (99.75) | − | − | yes (99.75) |
| PDC-7 (class C) | − | − | − | yes (99.75) | yes (99.75) | yes (99.75) | − | yes (98.99) | − | − |
| OXA-4 (class D) | − | − | − | − | − | − | − | − | yes (100) | − |
| OXA-10 (class D) | − | yes (99.62) | yes (100) | − | − | − | − | − | − | − |
| OXA-50 (class D) | yes (99.24) | yes (99.24) | yes (99.24) | yes (99.24) | yes (99.24) | yes (99.24) | yes (98.09) | yes (98.33) | yes (99.24) | yes (99.24) |
| Efflux pump regulators | ||||||||||
| MexR | + | + | + | + | + | + | + | + | + | + |
| NalC | + | + | + | + | + | + | + | + | + | + |
| NalD | + | + | + | + | + | + | + | + | + | + |
| CpxR | + | + | + | − | + | + | − | + | + | + |
| SoxR | + | + | + | − | + | + | − | − | + | + |
| type B NfxB | + | + | + | − | + | + | − | − | + | + |
| Efflux pump complex | ||||||||||
| MexAB-OprM | + | + | + | − | + | + | + | + | + | + |
| MexCD-OprJ | + | + | + | + | + | + | + | + | + | + |
| MexPQ-OpmE | + | + | + | + | + | + | + | + | + | + |
| MuxABC-OpmB | + | + | + | + | + | + | + | + | + | + |
| Antimicrobial susceptibility result (MIC in mg/L) | ||||||||||
| CZA | NS (128) | NS (96) | NS (12) | NS (24) | NS (16) | S (2) | S (1.5) | NS (24) | NS (32) | S (8) |
| C/T | NS (256) | NS (256) | NS (256) | NS (256) | NS (256) | NS (256) | NS (50) | NS (256) | NS (256) | NS (12) |
| MEM | NS (32) | NS (32) | NS (32) | NS (32) | S (2) | S (1.5) | S (0.75) | NS (32) | NS (32) | NS (32) |
| Mucoidity | non-mucoid | non-mucoid | non-mucoid | mucoid | non-mucoid | mucoid | non-mucoid | non-mucoid | mucoid | non-mucoid |
CZA, ceftazidime/avibactam; C/T, ceftolozane/tazobactam; MEM, meropenem; NS, non-susceptible; S, susceptible; +, present; −, absent.
Discussion
The impact of bacterial resistance on clinical outcomes and healthcare expenditure cannot be overstated.31 We found relatively high levels of non-susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in a clinical collection of MDR P. aeruginosa that pre-dated the introduction of these agents into clinical practice in Qatar. Moreover, ceftazidime/avibactam and ceftolozane/tazobactam activity was not consistent with each other or with other β-lactams. Susceptibility testing of P. aeruginosa isolates for ceftazidime/avibactam and ceftolozane/tazobactam is therefore essential for reliable clinical use.
The availability of ceftazidime/avibactam and ceftolozane/tazobactam as additional options for the treatment of MDR P. aeruginosa infections is a promising development. However, as noted in previous studies,17–19 their added value is limited by the observation that less than half of the isolates that were resistant to existing antipseudomonal β-lactam agents, aminoglycosides and quinolones were susceptible to either ceftazidime/avibactam or ceftolozane/tazobactam (Table 2). Unfortunately, the critical need for effective new treatment options for MDR P. aeruginosa remains to be met. The case for the importance of judicious clinical use has already been made by reports of rapid in vivo emergence of MDR P. aeruginosa resistance to ceftazidime/avibactam32 and ceftolozane/tazobactam33,34 in patients who received 10 days or less of treatment with the respective agent.
There are a few notable differences in our results compared with previous studies that compared ceftazidime/avibactam and ceftolozane/tazobactam activity against MDR P. aeruginosa recovered from patients without prior exposure to either agent (Table 4). The proportion of MDR P. aeruginosa isolates that were susceptible to ceftazidime/avibactam and/or ceftolozane/tazobactam was comparable to results reported in one previous study, but was considerably lower than in other reports (Table 4). This could be explained by the fact that the isolates included in those studies were generally less resistant.18–21 Moreover, unlike in this study, the majority of P. aeruginosa isolates in previous reports did not produce ESBLs or carbapenemases.18,19 Another possible explanation for this discrepancy is that different susceptibility testing methods were used. Whereas we used Liofilchem® MIC Test Strips, previous reports had used either broth microdilution17–19 or Etest.20,21 Previous investigators expressed concern that considerable proportions of their P. aeruginosa isolates had ceftazidime/avibactam MICs at the current CLSI breakpoint of 8 mg/L.17–19 In our study, a total of 82 (40.0%) of the isolates had ceftazidime/avibactam MICs within one doubling dilution of the breakpoint. Similarly, 40 (19.5%) isolates had ceftolozane/tazobactam MICs within one doubling dilution of the CLSI breakpoint of 4 mg/L.
Table 4.
Summary of studies comparing ceftazidime/avibactam and ceftolozane/tazobactam in vitro activity against MDR P. aeruginosa
| Study | Geographical location | Susceptibility testing method | Inclusion criteria | Collection years | Number included | Number (%) susceptible to MEM | Number (%) susceptible to CZA | Number (%) susceptible to C/T |
|---|---|---|---|---|---|---|---|---|
| Humphries et al.,17 2017 | Los Angeles, CA, USA | broth microdilution, except C/T by Etest | resistant to at least one antipseudomonal β-lactam antibiotic | 2015–16 | 309 | 49 (15.9) | 191 (61.8) | 224 (72.5) |
| Buehrle et al.,18 2016 | Pittsburgh, PA, USA | broth microdilution | MEM NS | not reported | 38 | 0 (0) | 35 (92.1) | 35 (92.1) |
| Grupper et al.,19 2017 | USA | broth microdilution | MEM NS | not reported | 290 | 0 (0) | 235 (81.0) | 264 (91.0) |
| Gonzalez et al.,20 2017 | St Louis, MO, USA | Etest | MEM NS | 2014 | 45 | 13 (28.9) | 37 (82.2) | 39 (86.7) |
| Alatoom et al.,21 2017 | Abu Dhabi, United Arab Emirates | Etest | resistant to at least one agent from at least three antimicrobial classes | 2015–16 | 31 | 15 (48.4) | 29 (93.5) | 30 (96.8) |
CZA, ceftazidime/avibactam; C/T, ceftolozane/tazobactam; MEM, meropenem; NS, non-susceptible.
All studies reported the isolates as susceptible if the MIC was ≤8 mg/L for ceftazidime/avibactam and ≤4 mg/L for ceftolozane/tazobactam.
In this study, 10 MDR P. aeruginosa isolates were subjected to WGS. Four isolates produced the class B carbapenemase VIM, the most common type of carbapenemase identified in P. aeruginosa isolates from the region.35,36 Half of the sequenced isolates produced ESBL enzymes (Table 3). Vietnamese ESBLs (VEB enzymes) are class A ESBLs that were originally described in P. aeruginosa isolates from South-East Asia.37 They are widely disseminated in P. aeruginosa from the Middle East,38–40 and have been associated with MDR P. aeruginosa outbreaks in Eastern Europe41,42 and in China.43 VEB enzymes are inhibited in vitro by avibactam, but result in resistance to ceftolozane/tazobactam.44,45
PDC enzymes, also known as AmpC, were identified in all of our sequenced isolates. Mutations leading to AmpC hyperproduction are amongst the most common mechanisms for β-lactam resistance in P. aeruginosa, including de novo and emergent resistance to ceftazidime/avibactam and ceftolozane/tazobactam.5,34,46–48 However, no such mutations were identified in any of the sequenced isolates in this study. Additionally, oxacillinases were detected in all sequenced isolates in this study. OXA-4, OXA-10 and OXA-50 are all narrow-spectrum β-lactamases.49,50 OXA-50, a naturally occurring β-lactamase, was present in all of the sequenced MDR P. aeruginosa included is this study. It was also described in previous reports of ceftazidime/avibactam- and/or ceftolozane/tazobactam-resistant MDR P. aeruginosa, without any evidence of mutation or overproduction.19,34,48 Interestingly, OXA-14, which is the product of a single point mutation in the OXA-10 gene, generates high-level resistance to ceftolozane/tazobactam in P. aeruginosa,33 while a 3 bp deletion in blaOXA-2 produced a novel enzyme, designated OXA-539, conferring high-level resistance to ceftazidime/avibactam.51
Unlike previous studies that compared the activity of ceftazidime/avibactam and ceftolozane/tazobactam against MDR P. aeruginosa, ceftazidime/avibactam was more active than ceftolozane/tazobactam in this study (Table 4). Focusing on the sequenced isolates offers a possible explanation for this observation. Isolates that were susceptible to ceftazidime/avibactam but not to ceftolozane/tazobactam (PA98, PA123 and PA154) produced β-lactamases belonging to class A, class C and class D, all of which are inhibited by avibactam, but not tazobactam. The VIM-producing isolates (PA9, PA37, PA125 and PA199) were, as expected, resistant to both ceftazidime/avibactam and ceftolozane/tazobactam. However, no immediate explanation is available for the remaining sequenced isolates (PA11 and PA12), which were non-susceptible to both agents without MBL production or detectable AmpC mutations.
The 10 sequenced MDR P. aeruginosa isolates belonged to six different STs. Three belonged to ST235, an international high-risk clone that has been associated with innumerable horizontally transferred resistance determinants.52,53 Others included the global ST233 and the Asian ST308 clones.53 These clones were previously reported from the Arabian Peninsula region, including from Qatar.36 This finding raises alarming concern of the potential for clonal dissemination of these high-risk multiresistant isolates and emphasizes the need to ensure the effective application of infection prevention and control measures.
To the best of our knowledge, this is the largest study from the Middle East comparing in parallel the in vitro activity of ceftazidime/avibactam and ceftolozane/tazobactam against MDR P. aeruginosa and investigating the possible underlying molecular mechanisms. A limitation of the current study is the use of Liofilchem® MIC Test Strips for ceftazidime/avibactam and ceftolozane/tazobactam susceptibility testing. Recent reports suggest that when compared to broth microdilution, this method can result in misclassification of some P. aeruginosa isolates as resistant to ceftazidime/avibactam and ceftolozane/tazobactam.54,55 Thus confirmation of our results using the broth microdilution reference method would have been ideal.
In conclusion, MDR P. aeruginosa susceptibility rates to ceftazidime/avibactam and ceftolozane/tazobactam were higher than those to all existing antipseudomonal agents, except colistin, but were less than 50% in extremely resistant isolates. Worryingly, MDR P. aeruginosa isolates from Qatar belonged to international high-risk clones and non-susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam was largely driven by the production of β-lactamases, including ESBL and VIM enzymes. Though ceftazidime/avibactam and ceftolozane/tazobactam offer opportunities to treat some patients with MDR P. aeruginosa, their extensive cross-resistance with other β-lactam agents implies that the need to continue to develop new agents, preferably with novel targets and mechanisms of action, remains as critical as ever.
Acknowledgements
We would like to thank Mr Jemal Hamid and Dr Devendra for technical support and Mr Mahmood Mohamed for statistical support.
Funding
The study was funded by an internal research grant (IRGC-01–51-033 to E. B. I.) from the Medical Research Centre at Hamad Medical Corporation, Doha, Qatar. Support was also provided by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas) (grant 219-2014-837 to J. J. and B. S.), the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) (Award Numbers R01AI100560, R01AI063517 and R01AI072219 to R. A. B.) and the Cleveland Department of Veterans Affairs (Award Number 1I01BX001974 to R. A. B.) from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development, and the Geriatric Research Education and Clinical Center VISN 10. The funders were not involved in the conduct of the study, the preparation of the manuscript or the decision to submit the manuscript for publication.
Transparency declarations
R. A. B. has received grants from Wockhardt, Merck, Entasis, Roche and GlaxoSmithKline. A. S. O. has received speakers’ honoraria from Pfizer, Merck and Gilead. All other authors: none to declare.
Author contributions
M. A. S. A., A. A. I. H. and S. A. J. conceived and designed the study and performed the experimental work. M. A. S. A., J. J. and A. S. O. analysed and interpreted the data. M. A. S. A., H. A. H., A. A. I. H. and A. S. O. prepared the manuscript. All authors critically reviewed the manuscript. All authors read and approved the final manuscript. All authors agreed on submission.
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of funders.
References
- 1. Morrissey I, Hackel M, Badal R. et al. A review of ten years of the Study for Monitoring Antimicrobial Resistance Trends (SMART) from 2002 to 2011. Pharmaceuticals (Basel) 2013; 6: 1335–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Weiner LM, Webb AK, Limbago B. et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2011-2014. Infect Control Hosp Epidemiol 2016; 37: 1288–301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Wisplinghoff H, Bischoff T, Tallent SM. et al. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 2004; 39: 309–17. [DOI] [PubMed] [Google Scholar]
- 4. Gellatly SL, Hancock R.. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 2013; 67: 159–73. [DOI] [PubMed] [Google Scholar]
- 5. López-Causapé C, Cabot G, del Barrio-Tofiño E. et al. The versatile mutational resistome of Pseudomonas aeruginosa. Front Microbiol 2018; 9: 685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Lister PD, Wolter DJ, Hanson ND.. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev 2009; 22: 582–610. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Zavascki AP, Carvalhaes CG, Picão RC. et al. Multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii: resistance mechanisms and implications for therapy. Expert Rev Anti Infect Ther 2010; 8: 71–93. [DOI] [PubMed] [Google Scholar]
- 8. Lodise TP, Patel N, Kwa A. et al. Predictors of 30-day mortality among patients with Pseudomonas aeruginosa bloodstream infections: impact of delayed appropriate antibiotic selection. Antimicrob Agents Chemother 2007; 51: 3510–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Peña C, Suarez C, Gozalo M. et al. Prospective multicenter study of the impact of carbapenem resistance on mortality in bloodstream infections. Antimicrob Agents Chemother 2012; 56: 1265–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Tam VH, Rogers CA, Chang K-T. et al. Impact of multidrug-resistant Pseudomonas aeruginosa bacteremia on patient outcomes. Antimicrob Agents Chemother 2010; 54: 3717–22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.WHO. Global Priority List of Antibiotic-resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics.https://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf? ua=1.
- 12. Liscio JL, Mahoney MV, Hirsch EB.. Ceftolozane/tazobactam and ceftazidime/avibactam: two novel β-lactam/β-lactamase inhibitor combination agents for the treatment of resistant Gram-negative bacterial infections. Int J Antimicrob Agents 2015; 46: 266–71. [DOI] [PubMed] [Google Scholar]
- 13. Ehmann DE, Jahic H, Ross PL. et al. Kinetics of avibactam inhibition against class A, C, and D β-lactamases. J Biol Chem 2013; 288: 27960–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Moya B, Zamorano L, Juan C. et al. Activity of a new cephalosporin, CXA-101 (FR264205), against β-lactam-resistant Pseudomonas aeruginosa mutants selected in vitro and after antipseudomonal treatment of intensive care unit patients. Antimicrob Agents Chemother 2010; 54: 1213–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Farrell DJ, Flamm RK, Sader HS. et al. Antimicrobial activity of ceftolozane-tazobactam tested against Enterobacteriaceae and Pseudomonas aeruginosa with various resistance patterns isolated in U.S. hospitals (2011-2012). Antimicrob Agents Chemother 2013; 57: 6305–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Sader HS, Farrell DJ, Castanheira M. et al. Antimicrobial activity of ceftolozane/tazobactam tested against Pseudomonas aeruginosa and Enterobacteriaceae with various resistance patterns isolated in European hospitals (2011-12). J Antimicrob Chemother 2014; 69: 2713–22. [DOI] [PubMed] [Google Scholar]
- 17. Humphries RM, Hindler JA, Wong-Beringer A. et al. Activity of ceftolozane-tazobactam and ceftazidime-avibactam against β-lactam-resistant Pseudomonas aeruginosa isolates. Antimicrob Agents Chemother 2017; 61: e01858–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Buehrle DJ, Shields RK, Chen L. et al. Evaluation of the in vitro activity of ceftazidime-avibactam and ceftolozane-tazobactam against meropenem-resistant Pseudomonas aeruginosa isolates. Antimicrob Agents Chemother 2016; 60: 3227–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Grupper M, Sutherland C, Nicolau DP.. Multicenter evaluation of ceftazidime-avibactam and ceftolozane-tazobactam inhibitory activity against meropenem-nonsusceptible Pseudomonas aeruginosa from blood, respiratory tract, and wounds. Antimicrob Agents Chemother 2017; 61: e00875–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Gonzalez MD, McMullen AR, Wallace MA. et al. Susceptibility of ceftolozane-tazobactam and ceftazidime-avibactam against a collection of β-lactam-resistant Gram-negative bacteria. Ann Lab Med 2017; 37: 174–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Alatoom A, Elsayed H, Lawlor K. et al. Comparison of antimicrobial activity between ceftolozane-tazobactam and ceftazidime-avibactam against multidrug-resistant isolates of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Int J Infect Dis 2017; 62: 39–43. [DOI] [PubMed] [Google Scholar]
- 22.State of Qatar Ministry of Public Health. National Health Strategy 2018-2022 https://www.moph.gov.qa/HSF/Documents/short%20report%20eng%2020.03.2018.pdf.
- 23. Magiorakos AP, Srinivasan A, Carey RB. et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012; 18: 268–81. [DOI] [PubMed] [Google Scholar]
- 24.Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing—Twenty-Ninth Edition: M100. CLSI, Wayne, PA, USA, 2019. [Google Scholar]
- 25. Bankevich A, Nurk S, Antipov D. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012; 19: 455–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Aziz RK, Bartels D, Best AA. et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics 2008; 9: 75.. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Larsen MV, Cosentino S, Lukjancenko O. et al. Benchmarking of methods for genomic taxonomy. J Clin Microbiol 2014; 52: 1529–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Camacho C, Coulouris G, Avagyan V. et al. BLAST+: architecture and applications. BMC Bioinformatics 2009; 10: 421.. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Larsen MV, Cosentino S, Rasmussen S. et al. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 2012; 50: 1355.. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Jia B, Raphenya AR, Alcock B. et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res 2017; 45: D566–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31. Mauldin PD, Salgado CD, Hansen IS. et al. Attributable hospital cost and length of stay associated with health care-associated infections caused by antibiotic-resistant gram-negative bacteria. Antimicrob Agents Chemother 2010; 54: 109–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Gangcuangco LM, Clark P, Stewart C. et al. Persistent bacteremia from Pseudomonas aeruginosa with in vitro resistance to the novel antibiotics ceftolozane-tazobactam and ceftazidime-avibactam. Case Rep Infect Dis 2016; 2016: 1520404.. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33. Fraile-Ribot PA, Cabot G, Mulet X. et al. Mechanisms leading to in vivo ceftolozane/tazobactam resistance development during the treatment of infections caused by MDR Pseudomonas aeruginosa. J Antimicrob Chemother 2018; 73: 658–63. [DOI] [PubMed] [Google Scholar]
- 34. Haidar G, Philips NJ, Shields RK. et al. Ceftolozane-tazobactam for the treatment of multidrug-resistant Pseudomonas aeruginosa infections: clinical effectiveness and evolution of resistance. Clin Infect Dis 2017; 65: 110–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35. Memish ZA, Assiri A, Almasri M. et al. Molecular characterization of carbapenemase production among gram-negative bacteria in Saudi Arabia. Microb Drug Resist 2015; 21: 307–14. [DOI] [PubMed] [Google Scholar]
- 36. Zowawi HM, Syrmis MW, Kidd TJ. et al. Identification of carbapenem-resistant Pseudomonas aeruginosa in selected hospitals of the Gulf Cooperation Council States: dominance of high-risk clones in the region. J Med Microbiol 2018; 67: 846–53. [DOI] [PubMed] [Google Scholar]
- 37. Poirel L, Naas T, Guibert M. et al. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum β-lactamase encoded by an Escherichia coli integron gene. Antimicrob Agents Chemother 1999; 43: 573–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38. Yezli S, Shibl AM, Memish ZA.. The molecular basis of β-lactamase production in Gram-negative bacteria from Saudi Arabia. J Med Microbiol 2015; 64: 127–36. [DOI] [PubMed] [Google Scholar]
- 39. Poirel L, Rotimi VO, Mokaddas EM. et al. VEB-1-like extended-spectrum β-lactamases in Pseudomonas aeruginosa, Kuwait. Emerg Infect Dis 2001; 7: 468–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40. Davodian E, Sadeghifard N, Ghasemian A. et al. Presence of blaPER-1 and blaVEB-1 β-lactamase genes among isolates of Pseudomonas aeruginosa from South West of Iran. J Epidemiol Glob Health 2016; 6: 211–3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41. Laudy AE, Róg P, Smolińska-Król K. et al. Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods. PLoS One 2017; 12: e0180121.. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42. Strateva T, Ouzounova-Raykova V, Markova B. et al. Widespread detection of VEB-1-type extended-spectrum β-lactamases among nosocomial ceftazidime-resistant Pseudomonas aeruginosa isolates in Sofia, Bulgaria. J Chemother 2007; 19: 140–5. [DOI] [PubMed] [Google Scholar]
- 43. Chen Z, Niu H, Chen G. et al. Prevalence of ESBLs-producing Pseudomonas aeruginosa isolates from different wards in a Chinese teaching hospital. Int J Clin Exp Med 2015; 8: 19400–5. [PMC free article] [PubMed] [Google Scholar]
- 44. Lahiri SD, Alm RA.. Identification of novel VEB β-lactamase enzymes and their impact on avibactam inhibition. Antimicrob Agents Chemother 2016; 60: 3183–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45. Ortiz de la Rosa J-M, Nordmann P, Poirel L.. ESBLs and resistance to ceftazidime/avibactam and ceftolozane/tazobactam combinations in Escherichia coli and Pseudomonas aeruginosa. J Antimicrob Chemother 2019; 74: 1934–9. [DOI] [PubMed] [Google Scholar]
- 46. Barnes MD, Taracila MA, Rutter JD. et al. Deciphering the evolution of cephalosporin resistance to ceftolozane-tazobactam in Pseudomonas aeruginosa. mBio 2018; 9: e02085-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47. Berrazeg M, Jeannot K, Ntsogo EV. et al. Mutations in β-lactamase AmpC increase resistance of Pseudomonas aeruginosa isolates to antipseudomonal cephalosporins. Antimicrob Agents Chemother 2015; 59: 6248–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48. MacVane SH, Pandey R, Steed LL. et al. Emergence of ceftolozane-tazobactam-resistant Pseudomonas aeruginosa during treatment is mediated by a single AmpC structural mutation. Antimicrob Agents Chemother 2017; 61: e01183-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49. Poirel L, Naas T, Nordmann P.. Diversity, epidemiology, and genetics of class D β-lactamases. Antimicrob Agents Chemother 2010; 54: 24–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50. Zhao WH, Hu ZQ.. β-Lactamases identified in clinical isolates of Pseudomonas aeruginosa. Crit Rev Microbiol 2010; 36: 245–58. [DOI] [PubMed] [Google Scholar]
- 51. Fraile-Ribot PA, Mulet X, Cabot G. et al. In vivo emergence of resistance to novel cephalosporin-β-lactamase inhibitor combinations through the duplication of amino acid D149 from OXA-2 β-lactamase (OXA-539) in sequence type 235 Pseudomonas aeruginosa. Antimicrob Agents Chemother 2017; 61: e01117-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52. Edelstein MV, Skleenova EN, Shevchenko OV. et al. Spread of extensively resistant VIM-2-positive ST235 Pseudomonas aeruginosa in Belarus, Kazakhstan, and Russia: a longitudinal epidemiological and clinical study. Lancet Infect Dis 2013; 13: 867–76. [DOI] [PubMed] [Google Scholar]
- 53. Oliver A, Mulet X, Lopez-Causape C. et al. The increasing threat of Pseudomonas aeruginosa high-risk clones. Drug Resist Update 2015; 21-22: 41–59. [DOI] [PubMed] [Google Scholar]
- 54. Humphries RM, Hindler JA, Magnano P. et al. Performance of ceftolozane-tazobactam Etest, MIC test strips, and disk diffusion compared to reference broth microdilution for β-lactam-resistant Pseudomonas aeruginosa isolates. J Clin Microbiol 2018; 56: e01633-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55. Schaumburg F, Bletz S, Mellmann A. et al. Comparison of methods to analyse susceptibility of German MDR/XDR Pseudomonas aeruginosa to ceftazidime/avibactam. Int J Antimicrob Agents 2019; 54: 255–60. [DOI] [PubMed] [Google Scholar]