Abstract
Objectives
To investigate the susceptibility of imipenem-non-susceptible Escherichia coli (INS-EC), Klebsiella pneumoniae (INS-KP), Acinetobacter baumannii (INS-AB) and Pseudomonas aeruginosa (INS-PA) to novel antibiotics.
Methods
MICs were determined using the broth microdilution method. Carbapenemase and ESBL phenotypic testing and PCR for genes encoding ESBLs, AmpCs and carbapenemases were performed.
Results
Zidebactam, avibactam and relebactam increased the respective susceptibility rates to cefepime, ceftazidime and imipenem of 17 INS-EC by 58.8%, 58.8% and 70.6%, of 163 INS-KP by 77.9%, 88.3% and 76.1% and of 81 INS-PA by 45.7%, 38.3% and 85.2%, respectively. Vaborbactam increased the meropenem susceptibility of INS-EC by 41.2% and of INS-KP by 54%. Combinations of β-lactams and novel β-lactamase inhibitors or β-lactam enhancers (BLI-BLE) were inactive against 136 INS-AB. In 58 INS-EC and INS-KP with exclusively blaKPC-like genes, zidebactam, avibactam, relebactam and vaborbactam increased the susceptibility of the partner β-lactams by 100%, 96.6%, 84.5% and 75.9%, respectively. In the presence of avibactam, ceftazidime was active in an additional 85% of 20 INS-EC and INS-KP with exclusively blaOXA-48-like genes while with zidebactam, cefepime was active in an additional 75%. INS-EC and INS-KP with MBL genes were susceptible only to cefepime/zidebactam. The β-lactam/BLI-BLE combinations were active against INS-EC and INS-KP without detectable carbapenemases. For INS-EC, INS-KP and INS-AB, tigecycline was more active than omadacycline and eravacycline but eravacycline had a lower MIC distribution. Lascufloxacin and delafloxacin were active in <35% of these INS isolates.
Conclusions
β-Lactam/BLI-BLE combinations were active in a higher proportion of INS-EC, INS-KP and INS-PA. The susceptibility of novel fluoroquinolones and tetracyclines was not superior to that of old ones.
Introduction
Antimicrobial resistance (AMR) is a major public health problem worldwide.1 In addition to worse patient outcome, AMR is associated with higher healthcare costs and productivity losses. In the EU, an estimated 33 110 deaths and 874 541 disability-adjusted life-years were attributable to AMR in 2015.2 Carbapenems have been successfully used to treat antibiotic-resistant Gram-negative pathogens but resistance emerges quickly. Among carbapenem-resistant Gram-negative pathogens, carbapenem-resistant Enterobacterales, Acinetobacter baumannii and Pseudomonas aeruginosa are of greater clinical importance due to their high prevalence, concomitant resistance to other antibiotics and negative impact on patient outcome. They are therefore on the priority list of bacteria for which new antibiotics are urgently needed.1
The assessment of the pipeline in 2019 by WHO revealed that 8 new antibiotics and combination agents gained market authorization between July 2017 and September 2019, with another 32 antibiotics targeting WHO priority pathogens in the Phase 1–3 development stage.3 Some of these recently developed antibiotics have shown good in vitro activity against drug-resistant pathogens.4–8 However, the demographic characteristics of patients from whom isolates were collected differed in each study, i.e. infection sites, healthcare settings or geographical area. These factors are associated with in vitro activity of antibiotics. For example, ceftolozane/tazobactam was less active against Gram-negative pathogens isolated in Vietnam than against those from other Asia-Pacific regions.8 In addition, the various inclusion criteria, such as the definition of carbapenem resistance or types of carbapenemase gene, also affect the susceptibility results. Avibactam has better inhibitory activity against OXA-48 compared with relebactam and vaborbactam but neither inhibit MBLs.4,5 In contrast, the activities of eravacycline and plazomicin are not affected by the types of carbapenemases per se.7 The interpretation of the susceptibility results of recently developed antibiotics from different studies is therefore difficult. This multicentre study concomitantly determined susceptibility to eight recently developed antibiotics of imipenem-non-susceptible pathogens from a nationwide surveillance programme in Taiwan and their activities were compared with commonly used antibiotics. Susceptibility was further analysed according to phenotypic and molecular characteristics.
Materials and methods
Ethics
The Taiwan Surveillance of Antimicrobial Resistance (TSAR) bacterial isolates were recovered from clinical samples taken as part of standard care and the TSAR project was approved by the Research Ethics Committee of the National Health Research Institutes (EC1010602-E, EC1030406-E and EC1050606-E).
Bacterial isolates
TSAR is a biennial longitudinal multicentre surveillance programme on clinical isolates.9 Isolates were stored frozen and subcultured onto appropriate agar plates for purity check before subsequent testing. Speciation was confirmed using conventional biochemical tests and API 20E, 32GN or VITEK II (bioMérieux, Marcy l’Étoile, France) as needed and A. baumannii was additionally identified using multiplex PCR.10 In this study, around 400 imipenem-non-susceptible isolates were selected, including Escherichia coli and Klebsiella pneumoniae with imipenem MIC ≥ 2 mg/L from 2012–18, and A. baumannii and P. aeruginosa with MIC ≥ 4 mg/L from 2018. The selection algorithm is shown in Figure S1, available as Supplementary data at JAC Online.
Antimicrobial susceptibility testing
The MICs of recently developed antibiotics were determined by broth microdilution using 96-well microtitre plates prepared in-house, following CLSI-recommended protocols.11 Recently developed antibiotics that we had access to included imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam, lascufloxacin, delafloxacin, eravacycline and omadacycline. Relebactam, vaborbactam, avibactam, eravacycline and omadacycline were obtained from MedChemExpress (USA), zidebactam and lascufloxacin were from MedKoo Biosciences (USA) and delafloxacin was from Sigma–Aldrich (USA). Relebactam, vaborbactam and avibactam were tested at fixed concentrations of 4, 8 and 4 mg/L, respectively, in combination with doubling dilutions of their partner β-lactams. Zidebactam was tested in combination with cefepime at a 1:1 ratio. The following quality control strains were included: E. coli ATCC 25922 (eravacycline, lascufloxacin and omadacycline) and P. aeruginosa ATCC 27853 (imipenem/relebactam, meropenem/vaborbactam, ceftazidime/avibactam, cefepime/zidebactam and delafloxacin).
MICs of cefepime, ceftazidime, ciprofloxacin, levofloxacin, imipenem, meropenem, minocycline and tigecycline that were meant to be compared with the aforementioned antibiotics were also determined using 96-well microtitre plates prepared in-house. MICs of other commonly used antibiotics were determined using custom-designed NHRIGN8, NHRIGN9 or NHRIGN10 panels prepared by Sensititre (Trek Diagnostics, West Sussex, UK) for E. coli and K. pneumoniae and standard GNX3F panels for A. baumannii and P. aeruginosa. E. coli ATCC 25922, P. aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used as quality control strains.
PCR screening for β-lactamase genes
Multiplex PCRs12–14 were performed on imipenem-non-susceptible isolates to detect the genes encoding class A (blaNMC, blaSME, blaIMI, blaKPC and blaGES), class B (blaIMP, blaVIM, blaGIM, blaSPM, blaSIM-1 and blaNDM) and class D (blaOXA-48-like, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like and blaOXA-51-like) β-lactamases with carbapenemase activity (Figure S1). E. coli and K. pneumoniae isolates that were negative for carbapenemase genes were subjected to PCR targeting genes encoding ESBLs (blaSHV as well as blaCTX-M-group-1, -2, -8, -9 and -25) and AmpCs (blaMOX, blaCIT, blaDHA, blaACC, blaMIR and blaFOX).15–18 The blaSHV amplicon was further digested by restriction endonuclease NheІ to identify the presence of Gly238Ser in ESBLs.16
Phenotypic testing
The modified carbapenem inactivation method (mCIM) and the EDTA-modified carbapenem inactivation method (eCIM) were performed on E. coli and K. pneumoniae to detect the activity of serine- and metal-dependent carbapenemases. Isolates without carbapenemase production were further subjected to ESBL phenotypic testing (Figure S1). The experimental procedures were in accordance with CLSI recommendations.11
Data analysis
Susceptibilities were calculated using Whonet software (Stelling and O’Brien).9 The interpretation of susceptibility was in accordance with 2019 CLSI criteria,19 and US FDA criteria if CLSI breakpoints were not available. The breakpoints of novel antibiotics used in this study are listed in Table S1. For comparison with cefepime/zidebactam (susceptible ≤8/8 mg/L), the susceptible dose-dependent (SDD) and susceptible breakpoints (≤8 mg/L) for cefepime were used in this study. Due to the lack of tigecycline MIC breakpoints for Acinetobacter spp., the US FDA breakpoints for Enterobacterales were used (≤2 mg/L susceptible; >4 mg/L resistant). The breakpoints of colistin followed 2019 EUCAST recommendations (≤2 mg/L susceptible; >2 mg/L resistant).
Results
Clinical characteristics
Among the 6806 E. coli and 3021 K. pneumoniae isolates from 2012 and 2018, and the 191 A. baumannii and 557 P. aeruginosa isolates from 2018, the numbers of isolates that were non-susceptible to imipenem were 17 (0.2%), 163 (5.4%), 136 (71.2%) and 81(14.5%), respectively (Figure S1). The clinical characteristics of patients from whom these isolates were recovered are listed in Table S2. These isolates were mostly from adults, in non-ICU wards and in regional hospitals but the specimen sites and hospital geographical regions varied.
Antimicrobial susceptibility of imipenem-non-susceptible E. coli and K. pneumoniae
Table 1 shows the antimicrobial susceptibility of imipenem-non-susceptible E. coli and K. pneumoniae. Both bacteria were highly resistant to commonly used non-carbapenem β-lactam antibiotics. Even piperacillin/tazobactam, which exerted the highest inhibitory effect, inhibited only 17.6% and 9.8% of imipenem-non-susceptible E. coli and K. pneumoniae, respectively. The addition of novel β-lactamase inhibitors or β-lactam enhancers (BLI-BLE) greatly increased the susceptibility of β-lactam antibiotics. Zidebactam, avibactam, relebactam and vaborbactam increased the susceptibility to cefepime, ceftazidime, imipenem and meropenem of E. coli by 58.8% (100% versus 41.2%), 58.8% (70.6% versus 11.8%), 70.6% (70.6% versus 0%) and 41.2% (94.1% versus 52.9%) and of K. pneumoniae by 77.9% (99.4% versus 21.5%), 88.3% (90.8% versus 2.5%), 76.1% (76.1% versus 0%) and 54.0% (81.6% versus 27.6%), respectively. Concurrently, there was a consistent left shift of MIC distribution for these β-lactam antibiotics in the presence of the novel BLI-BLEs (Table S3).
Table 1.
Antimicrobial susceptibility of imipenem-non-susceptible E. coli and K. pneumoniae from the TSAR programme, 2012–18
|
E. coli (n = 17) |
K. pneumoniae (n = 163) |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Antibiotic | R (%) | I (%) | S (%) | MIC50 (mg/L) | MIC90 (mg/L) | MIC range (mg/L) | R (%) | I (%) | S (%) | MIC50 (mg/L) | MIC90 (mg/L) | MIC range (mg/L) |
| Ampicillin | 100 | 0 | 0 | >16 | >16 | >16 | 100 | 0 | 0 | >16 | >16 | >16 |
| Cefazolin | 100 | 0 | 0 | >16 | >16 | >16 | 99.4 | 0 | 0.6 | >16 | >16 | 4 to >16 |
| Cefepimea | 58.8 | 0 | 41.2 | 16 | >32 | ≤0.25 to >32 | 78.5 | 0 | 21.5 | >32 | >32 | ≤0.25 to >32 |
| Cefepime/ zidebactam | 0 | 0 | 100 | 1 | 4 | ≤0.25–4 | 0.6 | 0 | 99.4 | 1 | 4 | ≤0.25 to >32 |
| Cefotaxime | 94.1 | 0 | 5.9 | >32 | >32 | ≤1 to >32 | 96.3 | 1.2 | 2.5 | >32 | >32 | ≤1 to >32 |
| Cefoxitin | 94.1 | 5.9 | 0 | >16 | >16 | 16 to >16 | 98.2 | 0 | 1.8 | >16 | >16 | ≤4 to >16 |
| Ceftazidime | 88.2 | 0 | 11.8 | >32 | >32 | ≤0.25 to >32 | 96.3 | 1.2 | 2.5 | >32 | >32 | ≤0.25 to >32 |
| Ceftazidime/ avibactam | 29.4 | 0 | 70.6 | 2 | >32 | ≤0.25 to >32 | 9.2 | 0 | 90.8 | 2 | 8 | ≤0.25 to >32 |
| Cefuroxime | 100 | 0 | 0 | >16 | >16 | >16 | 97.5 | 0 | 2.5 | >16 | >16 | 8 to >16 |
| Imipenem | 47.1 | 52.9 | 0 | 2 | >16 | 2 to >16 | 71.2 | 28.8 | 0 | 8 | >16 | 2 to >16 |
| Imipenem/ relebactam | 29.4 | 0 | 70.6 | 1 | 8 | ≤0.125 to >16 | 16 | 8 | 76.1 | 0.5 | 4 | ≤0.125–16 |
| Meropenem | 35.3 | 11.8 | 52.9 | 1 | >32 | 0.125 to >32 | 67.5 | 4.9 | 27.6 | 8 | >32 | ≤0.06 to >32 |
| Meropenem/ vaborbactam | 5.9 | 0 | 94.1 | 0.5 | 4 | ≤0.06 to >32 | 6.7 | 11.7 | 81.6 | 2 | 8 | ≤0.06 to >32 |
| Piperacillin/ tazobactam | 70.6 | 11.8 | 17.6 | >64 | >64 | ≤4 to >64 | 87.1 | 3.1 | 9.8 | >64 | >64 | ≤4 to >64 |
| Ciprofloxacin | 82.4 | 0 | 17.6 | >16 | >16 | ≤0.03 to >16 | 91.4 | 4.9 | 3.7 | >16 | >16 | ≤0.03 to >16 |
| Delafloxacin | 76.5 | 0 | 23.5 | 8 | >32 | ≤0.06 to >32 | 97.5 | 0 | 2.5 | >32 | >32 | ≤0.06 to >32 |
| Lascufloxacin | 76.5 | 0 | 23.5 | 32 | >64 | 0.06 to >64 | 96.3 | 1.8 | 1.8 | >64 | >64 | 0.125 to >64 |
| Levofloxacin | 82.4 | 0 | 17.6 | 32 | >32 | ≤0.25 to >32 | 89 | 3.7 | 7.4 | >32 | >32 | ≤0.25 to >32 |
| Eravacycline | 23.5 | 0 | 76.5 | 0.5 | 1 | 0.125–2 | 63.2 | 0 | 36.8 | 1 | 2 | 0.25 to >8 |
| Minocycline | 23.5 | 11.8 | 64.7 | 4 | 16 | 1–32 | 27.6 | 42.9 | 29.4 | 8 | 32 | 1 to >64 |
| Omadacycline | 11.8 | 17.6 | 70.6 | 4 | 16 | 1–32 | 27.6 | 44.2 | 28.2 | 8 | 16 | 2 to >32 |
| Tigecycline | 0 | 0 | 100 | 0.5 | 2 | 0.25–2 | 0.6 | 2.5 | 96.9 | 1 | 2 | 0.25–8 |
| Amikacin | 5.9 | 0 | 94.1 | ≤4 | 16 | ≤4 to >32 | 22.7 | 0.6 | 76.7 | ≤4 | >32 | ≤4 to >32 |
| Gentamicin | 41.2 | 0 | 58.8 | 2 | >8 | ≤1 to >8 | 66.9 | 1.2 | 31.9 | >8 | >8 | ≤1 to >8 |
| Aztreonam | 88.2 | 5.9 | 5.9 | >16 | >16 | ≤1 to >16 | 86.5 | 4.9 | 8.6 | >16 | >16 | ≤1 to >16 |
| Colistin | 5.9 | 0 | 94.1 | ≤0.5 | ≤0.5 | ≤0.5 to >2 | 12.9 | 0 | 87.1 | ≤0.5 | >2 | ≤0.5 to >2 |
R, resistant; I intermediate; S, susceptible.
SDD and susceptible breakpoints (≤8 mg/L) for cefepime were used here.
Imipenem-non-susceptible E. coli and K. pneumoniae had similar high rates of non-susceptibility to ciprofloxacin and levofloxacin and the newer fluoroquinolones lascufloxacin and delafloxacin (Table 1). Susceptibility to these fluoroquinolones ranged from 17.6% to 23.5% in E. coli and was even lower (<10%) in K. pneumoniae. Tigecycline was the most active tetracycline tested (>95% susceptibility) but the MIC range of eravacycline was similar to or lower than that of tigecycline (Table S3 and Figure S2). The rates of susceptibility to minocycline, eravacycline and omadacycline were 64.7%, 76.5% and 70.6% in E. coli but were only 29.4%, 36.8% and 28.2%, respectively, in K. pneumoniae.
Effect of carbapenemase genotypes and phenotypes on susceptibility
The blaOXA-48-like (n = 2), blaVIM-like (1), blaIMP-like (1) and blaNDM-like (1) genes were found in 5 of the 17 E. coli (29.4%). Among 87 of the 163 K. pneumoniae harbouring carbapenemase genes (53.4%), blaKPC-like (n = 62) was the most commonly identified gene, followed by blaOXA-48-like (19), blaIMP-like (5), blaVIM-like (5) and blaNDM-like (1). The low prevalence of carbapenemases in imipenem-non-susceptible E. coli and K. pneumoniae in Taiwan has been reported.20,21 Among the 88 E. coli and K. pneumoniae isolates without any detectable carbapenemase gene, 85 had ampC and/or blaESBL.
None of the 58 K. pneumoniae and E. coli isolates with blaKPC-like genes was susceptible to cefepime, ceftazidime, imipenem or meropenem but zidebactam, avibactam, relebactam and vaborbactam restored their susceptibility to 100%, 96.6%, 84.5% and 75.9%, respectively (Table 2). Avibactam and zidebactam increased the susceptibility of isolates with blaOXA-48-like genes to ceftazidime and cefepime by 85% and 75%, respectively. Intriguingly, the addition of relebactam increased imipenem susceptibility in six isolates with blaOXA-48-like genes, whose MICs were marginally decreased by 2–4-fold. The β-lactam/novel BLI-BLE combinations were highly active in vitro against isolates without detectable carbapenemases regardless of the presence of ampC and/or blaESBL.
Table 2.
Susceptibility of imipenem-non-susceptible bacteria with different genotypes and phenotypes to β-lactams with and without novel BLI-BLEs
| Species with different genotypes and phenotypes | N | Susceptible (%) |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| FEPa | FPZ | CAZ | CZA | IPM | IMR | MEM | MEV | ||
| E. coli and K. pneumoniae | 180 | 23.3 | 99.4 | 3.3 | 88.9 | 0 | 75.6 | 30 | 82.8 |
| genotype | |||||||||
| with carbapenemase gene | 92 | 5.4 | 100 | 2.2 | 82.6 | 0 | 59.8 | 7.6 | 70.7 |
| with class B carbapenemase gene | 13 | 0 | 100 | 0 | 0 | 0 | 0 | 7.7 | 53.8 |
| with blaKPC-like only | 58 | 0 | 100 | 0 | 96.6 | 0 | 84.5 | 0 | 75.9 |
| with blaOXA-48-like only | 20 | 25 | 100 | 10 | 95 | 0 | 30 | 30 | 70 |
| without carbapenemase gene | 88 | 42 | 98.9 | 4.5 | 95.5 | 0 | 92 | 53.4 | 95.5 |
| with ESBL genea only | 1 | 0 | 100 | 100 | 100 | 0 | 100 | 100 | 100 |
| with AmpC gene only | 41 | 85.4 | 97.6 | 2.4 | 95.1 | 0 | 95.1 | 63.4 | 95.1 |
| with ESBL and AmpC geneb | 43 | 0 | 100 | 0 | 95.3 | 0 | 90.7 | 41.9 | 95.3 |
| phenotypec | |||||||||
| class B carbapenemase producerd | 11 | 0 | 100 | 0 | 0 | 0 | 0 | 9.1 | 45.5 |
| non-MBL producer | 82 | 7.3 | 100 | 2.4 | 93.9 | 0 | 68.3 | 8.5 | 74.4 |
| non-carbapenemase producer | 87 | 41.4 | 98.9 | 4.6 | 95.4 | 0 | 92 | 52.9 | 95.4 |
| ESBL producer | 22 | 4.5 | 100 | 4.5 | 100 | 0 | 100 | 72.7 | 100 |
| A. baumannii | 136 | 1.5 | 8.1 | 2.2 | 1.5 | 0 | 0.7 | 0 | 0 |
| genotype | |||||||||
| with class D carbapenemase gene | 135 | 1.5 | 8.1 | 2.2 | 1.5 | 0 | 0 | 0 | 0 |
| blaOXA-23-like | 117 | 0 | 6.8 | 1.7 | 0.9 | 0 | 0 | 0 | 0 |
| blaOXA-24-like | 22 | 4.5 | 13.6 | 0 | 4.5 | 0 | 0 | 0 | 0 |
| P. aeruginosa | 81 | 45.7 | 91.4 | 43.2 | 81.5 | 0 | 85.2 | 28.4 | 42.0 |
| genotype | |||||||||
| with class B carbapenemase gene | 4 | 0 | 75 | 0 | 0 | 0 | 0 | 0 | 0 |
| without carbapenemase gene | 77 | 48.1 | 92.2 | 45.5 | 85.7 | 0 | 89.6 | 29.9 | 44.2 |
FEP, cefepime; FPZ, cefepime/zidebactam; CAZ, ceftazidime; CZA, ceftazidime/avibactam; IPM; imipenem; IMR, imipenem/relebactam; MEM, meropenem; MEV, meropenem/vaborbactam.
SDD and susceptible breakpoints (≤8 mg/L) for cefepime were used here.
Only prevalent ESBL genes were tested.
Isolates with positive mCIM and eCIM were defined as class B carbapenemase (MBL) producers; those with positive mCIM but negative eCIM were non-MBL carbapenemase producers; those with negative mCIM were non-carbapenemase producers.
Seven of the 18 isolates positive for both eCIM and mCIM were only positive for blaKPC-like by PCR, which was further confirmed by WGS. Therefore, these seven isolates were categorized as non-MBL carbapenemase producers.
The types of carbapenemases identified by phenotypic testing were in accordance with the PCR except seven K. pneumoniae isolates that were falsely categorized as class B carbapenemase producers by the phenotypic testing. They were excluded from the class B carbapenemase producers because WGS showed only the presence of blaKPC-like genes and they were susceptible to β-lactam/novel BLI-BLE combinations (data not shown). After re-categorizing these seven isolates into the non-MBL-producer group, the effect of phenotypes on susceptibility to the β-lactam/novel BLI-BLE combinations was also similar to the observed effect of genotypes (Table 2). Susceptibility to fluoroquinolones and tetracyclines, either new or old agents, was not contingent on the genotypes and phenotypes of carbapenemase (Table S4) and susceptibility to tigecycline and colistin remained at >80% in all subgroups.
Antimicrobial susceptibility in imipenem-non-susceptible A. baumannii
The 136 imipenem-non-susceptible A. baumannii isolates were highly resistant to all β-lactams, with or without BLI-BLE, as well as all fluoroquinolones tested, with susceptibility rates of less than 10% (Table 3). Among tetracyclines, tigecycline was the most active, followed by minocycline, omadacycline and eravacycline (susceptibility rates of 70.6%, 40.4%, 36% and 19.1%, respectively). However, the MIC distribution, MIC50 and MIC90 were the lowest for eravacycline, followed by tigecycline (Table 3 and Table S3).
Table 3.
Antimicrobial susceptibility of imipenem-non-susceptible A. baumannii and P. aeruginosa from the TSAR programme, 2018
| Antibiotics |
A. baumannii (n = 136) |
P. aeruginosa (n = 81) |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| R (%) | I (%) | S (%) | MIC50 (mg/L) | MIC90 (mg/L) | MIC range (mg/L) | R (%) | I (%) | S (%) | MIC50 (mg/L) | MIC90 (mg/L) | MIC range (mg/L) | |
| Ampicillin/ sulbactam | 86.8 | 11.8 | 1.5 | 64 | >64 | ≤4 to >64 | Intrinsic resistance | |||||
| Cefepime | 97.1 | 1.5 | 1.5 | >32 | >32 | 8 to >32 | 28.4 | 25.9 | 45.7 | 16 | 32 | 1 to >32 |
| Cefepime/ zidebactam | 38.2 | 53.7 | 8.1 | 16 | 32 | 8 to >32 | 0 | 8.6 | 91.4 | 4 | 8 | 0.5–16 |
| Cefotaxime | 96.3 | 3.7 | 0 | >32 | >32 | 16 to >32 | Intrinsic resistance | |||||
| Ceftazidime | 97.1 | 0.7 | 2.2 | >32 | >32 | 4 to >32 | 44.4 | 12.3 | 43.2 | 16 | >32 | 1 to >32 |
| Ceftazidime/ avibactam | 98.5 | 0 | 1.5 | >32 | >32 | 8 to >32 | 18.5 | 0 | 81.5 | 4 | 32 | 1 to >32 |
| Imipenem | 99.3 | 0.7 | 0 | >16 | >16 | 4 to >16 | 70.4 | 29.6 | 0 | 16 | >16 | 4 to >16 |
| Imipenem/ relebactam | 99.3 | 0 | 0.7 | >16 | >16 | 1 to >16 | 7.4 | 7.4 | 85.2 | 1 | 4 | 0.25–16 |
| Meropenem | 99.3 | 0.7 | 0 | >32 | >32 | 4 to >32 | 61.7 | 9.9 | 28.4 | 8 | 32 | 0.125 to >32 |
| Meropenem/ vaborbactam | 98.5 | 1.5 | 0 | >32 | >32 | 8 to >32 | 35.8 | 22.2 | 42 | 8 | 32 | 0.25 to >32 |
| Piperacillin/ tazobactam | 98.5 | 1.5 | 0 | >64 | >64 | 32 to >64 | 29.6 | 19.8 | 50.6 | 16 | >64 | ≤8 to >64 |
| Ciprofloxacin | 99.3 | 0 | 0.7 | >16 | >16 | 1 to >16 | 56.8 | 7.4 | 35.8 | 2 | >16 | 0.06 to >16 |
| Delafloxacin | 98.5 | 0 | 1.5 | 4 | 16 | 0.25 to >32 | 56.8 | 12.3 | 30.9 | 2 | 32 | 0.125 to >32 |
| Lascufloxacin | 79.4 | 18.4 | 2.2 | 8 | 16 | 1 to >64 | 97.5 | 2.5 | 0 | 64 | >64 | 2 to >64 |
| Levofloxacin | 93.4 | 5.9 | 0.7 | 16 | >32 | ≤0.25 to >32 | 65.4 | 6.2 | 28.4 | 4 | >32 | ≤0.25 to >32 |
| Eravacycline | 80.9 | 0 | 19.1 | 1 | 2 | 0.125–8 | Intrinsic resistance | |||||
| Minocycline | 17.6 | 41.9 | 40.4 | 8 | 16 | ≤0.5–16 | Intrinsic resistance | |||||
| Omadacycline | 8.1 | 55.9 | 36 | 8 | 8 | 1–32 | Intrinsic resistance | |||||
| Tigecycline | 6.6 | 22.8 | 70.6 | 2 | 4 | 0.25–16 | Intrinsic resistance | |||||
| Amikacin | 88.2 | 0.7 | 11 | >32 | >32 | ≤4 to >32 | 2.5 | 2.5 | 95 | ≤4 | 8 | ≤4 to >32 |
| Gentamicin | 90.4 | 2.9 | 6.6 | >8 | >8 | 1 to >8 | 19.8 | 1.2 | 79 | 2 | >8 | ≤1 to >8 |
| Tobramycin | 89 | 2.2 | 8.8 | >8 | >8 | ≤1 to >8 | 19.8 | 0 | 80.2 | ≤1 | >8 | ≤1 to >8 |
| Aztreonam | Intrinsic resistance | 33 | 13.6 | 53.1 | 8 | >16 | ≤2 to >16 | |||||
| Colistin | 0.7 | 0 | 99.3 | ≤0.25 | 0.5 | ≤0.25 to >4 | 3.7 | 0 | 96 | 1 | 2 | ≤0.25 to >4 |
R, resistant; I intermediate; S, susceptible.
The blaOXA-23-like, blaOXA-24-like, ISAba1-blaOXA-51-like and blaOXA-58-like genes were found in 117, 22, 11 and 1 isolates, respectively. In isolates with blaOXA-23-like or blaOXA-24-like genes, susceptibility to the β-lactam/novel BLI-BLE combinations, as well as newly developed fluoroquinolones and tetracyclines, remained similar to the overall trend (Table 2 and Table S4).
Antimicrobial susceptibility in imipenem-non-susceptible P. aeruginosa
The susceptibility rates to many commonly used broad-spectrum β-lactams, i.e. aztreonam, cefepime, ceftazidime and piperacillin/tazobactam, of imipenem-non-susceptible P. aeruginosa were around 40%–60%; only a few isolates (15 of 81; 18.5%) were resistant concomitantly to these commonly used broad-spectrum β-lactams. The addition of a novel BLI-BLE increased the susceptibility by 45.7%, 38.3% and 85.2% for cefepime, ceftazidime and imipenem, respectively, with a corresponding left shift in MICs (Table 3 and Table S3). In contrast, the addition of vaborbactam only marginally increased the meropenem susceptibility from 28.4% to 42%, without an MIC shift. Similar rates of susceptibility were observed to the new fluroquinolone delafloxacin and old fluoroquinolones (<40%) but no isolate was susceptible to lascufloxacin.
Most imipenem-non-susceptible P. aeruginosa isolates (77 of 81) lacked carbapenemase genes and were susceptible to β-lactam/novel BLI-BLE combinations, except meropenem/vaborbactam. Three of the four P. aeruginosa with blaVIM-like genes were susceptible to cefepime/zidebactam (Table 2), with an MIC decrease of 2–4-fold. Overall, susceptibility rates for amikacin and colistin were the highest.
Discussion
This study compared the in vitro activity of recently developed antibiotics with commonly used ones against four WHO priority bacteria. The addition of novel BLI-BLEs to β-lactams greatly increased their effect on imipenem-non-susceptible E. coli and K. pneumoniae; the degree of change depended on β-lactamase phenotypes and genotypes. Imipenem-non-susceptible A. baumannii were highly resistant to the β-lactam/novel BLI-BLEs tested. Cefepime/zidebactam, ceftazidime/avibactam and imipenem/relebactam had >80% inhibitory effect on imipenem-non-susceptible P. aeruginosa. Omadacycline, delafloxacin and lascufloxacin were not superior to commonly used antibiotics against imipenem-non-susceptible bacteria.
The activity of β-lactams in combination with novel BLI-BLEs varied depending on the resistance mechanisms in E. coli and K. pneumoniae. Our results were generally in accordance with previous studies.4,5 All new BLI-BLEs increased the susceptibility of partner β-lactams in isolates with blaKPC-like, blaESBL and/or ampC genes. Ceftazidime/avibactam was additionally active against isolates with blaOXA-48-like genes. However, meropenem/vaborbactam, which lacks activity against class B or D carbapenemase,22 appeared to inhibit isolates with these genes in our study (Table 2). The difference in breakpoints used for meropenem/vaborbactam (S ≤ 4 mg/L) and meropenem (S ≤ 1 mg/L) was the main factor responsible for the increased susceptibility since the MIC distributions of meropenem and meropenem/vaborbactam were similar in our isolates with class B or D carbapenemase genes (data not shown). A similar situation was also observed for P. aeruginosa (Table S1).
Cefepime/zidebactam was reported to be active against various pathogens harbouring class A, B or D carbapenemase genes.23–25 The effect was also observed in our E. coli and K. pneumoniae as well as P. aeruginosa with different resistance mechanisms. It is postulated that zidebactam promotes cefepime killing by targeting different PBPs but not inhibition of MBL.23–25 Other studies further showed its modest effect on A. baumannii; one study reported that the addition of zidebactam increased the proportion of isolates with cefepime MICs of ≤8 mg/L from 3.8% to 25.6%.6 However, the addition of zidebactam did not significantly lower the cefepime MICs to ≤8 mg/L in our A. baumannii isolates (Table S3).
Sfeir et al.26 demonstrated that eCIM had a sensitivity of 100% but a specificity of 90%–100%, due to two K. pneumoniae isolates with blaOXA-232 that tested positive initially but negative upon repeat testing. Our study showed that seven K. pneumoniae isolates with blaKPC-like genes repeatedly tested positive on eCIM under the same conditions.26 Using PCR as a standard, the sensitivity and specificity of eCIM were 100% (11/11) and 91.5% (75/82), respectively, which are similar to those shown by Sfeir et al. The eCIM had high accuracy but a small chance of false positivity is still possible.
Omadacycline, delafloxacin and lascufloxacin are indicated against Gram-positive pathogens and community-acquired infections since their activity against healthcare-associated Gram-negative pathogens was not expected to be superior to commonly used comparators, i.e. levofloxacin or tigecycline, as shown by previous studies and ours.27–29 Comparison of eravacycline and tigecycline activity was difficult due to different breakpoints set by the US FDA (≤0.5 and ≤2 mg/L, respectively), which resulted in lower susceptibility to eravacycline despite having lower MICs compared with tigecycline MICs seen in our isolates (Table S3 and Figure S2) and in previous studies.30 It is further complicated by the different breakpoints set by the US FDA and EUCAST. An agreement on breakpoints via harmonization of various regulatory agencies would facilitate future comparative studies.
There were limitations of our study. First, not all recently developed antibiotics were included due to the difficulty of access to drugs under development. Second, some isolates were carbapenemase negative so porin alteration and/or efflux pump overexpression likely played a role in these isolates.21 In Taiwan, porin loss in combination with the presence of AmpC or ESBL has been reported to be the main mechanism of carbapenem resistance in K. pneumoniae and E. coli.31 However, the roles of efflux pumps or porins were not determined in our study. Third, our PCR only targeted a limited number of genes, i.e. those encoding prevalent ESBLs (blaCTX-M-type but not blaTEM).21,32,33 Resistance mechanisms identified by WGS would be more comprehensive. Fourth, our study did not select for certain resistance mechanisms, i.e. class B carbapenemases; therefore, the number of isolates with a specific resistance mechanism was limited. However, our results for these isolates were still in concordance with previously published studies.4–8
In conclusion, β-lactam plus novel BLI-BLE combinations inhibited most imipenem-non-susceptible E. coli, K. pneumoniae and P. aeruginosa but none was active against imipenem-non-susceptible A. baumannii. Cefepime/zidebactam was the most active, even against isolates with an MBL. New fluoroquinolones and tetracyclines were not superior to old ones but eravacycline had lower MICs compared with tigecycline.
Supplementary Material
Acknowledgements
We express our sincere appreciation to the following hospitals for their participation in the TSAR: Buddhist Tzu Chi General Hospital; Cathay General Hospital; Changhua Christian Hospital; Cheng-Ching Hospital; Chung Shan Medical University Hospital; Da Chien General Hospital; Ditmanson Medical Foundation Chia-Yi Christian Hospital; Far Eastern Memorial Hospital; Hua-Lien Hospital; Jen-Ai Hospital; Kaohsiung Armed Forces General Hospital; Kaohsiung Chang Gung Memorial Hospital of the Chang Gung Medical Foundation; Kaohsiung Medical University Chung-Ho Memorial Hospital; Kaohsiung Veterans General Hospital; Kuang Tien General Hospital; Lo-Hsu Foundation, Inc., Lotung Poh-Ai Hospital; Mennonite Christian Hospital; Min-Sheng Healthcare; National Cheng Kung University Hospital; Saint Mary’s Hospital Luodong; Show Chwan Memorial Hospital; Tungs’ Taichung MetroHarbor Hospital; Taichung Veterans General Hospital; Tainan Sin-Lau Hospital, the Presbyterian Church in Taiwan; Taipei City Hospital Heping Fuyou Branch; Taipei City Hospital Zhongxiao Branch; Taipei Veterans General Hospital; and Tri-Service General Hospital.
Funding
This project was supported by an intramural grant from the National Health Research Institutes (IV-108-PP-09, IV-107-PP-09, IV-107-SP-01, IV-108-01) and the Ministry of Science and Technology (107-2320-B-400 -010 -MY3 and 109–2321-B-415 -004 -).
Transparency declarations
None to declare.
Supplementary data
Tables S1 to S4 and Figures S1 and S2 are available as Supplementary data at JAC Online.
References
- 1.WHO. WHO publishes list of bacteria for which new antibiotics are urgently needed. 2017. https://www.who.int/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed.
- 2.Cassini A, Hogberg LD, Plachouras D. et al. Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis. Lancet Infect Dis 2019; 19: 56–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.WHO. 2019 antibacterial agents in clinical development: an analysis of the antibacterial clinical development pipeline. 2019. https://www.who.int/publications/i/item/9789240000193.
- 4.Zhanel GG, Lawrence CK, Adam H. et al. Imipenem-relebactam and meropenem-vaborbactam: two novel carbapenem-β-lactamase inhibitor combinations. Drugs 2018; 78: 65–98. [DOI] [PubMed] [Google Scholar]
- 5.Zasowski EJ, Rybak JM, Rybak MJ.. The β-lactams strike back: ceftazidime-avibactam. Pharmacotherapy 2015; 35: 755–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Khan Z, Iregui A, Landman D. et al. Activity of cefepime/zidebactam (WCK 5222) against Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii endemic to New York City medical centres. J Antimicrob Chemother 2019; 74: 2938–42. [DOI] [PubMed] [Google Scholar]
- 7.Sheu CC, Chang YT, Lin SY. et al. Infections caused by carbapenem-resistant Enterobacteriaceae: an update on therapeutic options. Front Microbiol 2019; 10: 80. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Kuo SC, Liu CE, Lu PL. et al. Activity of ceftolozane-tazobactam against Gram-negative pathogens isolated from lower respiratory tract infections in the Asia-Pacific region: SMART 2015-2016. Int J Antimicrob Agents 2020; 55: 105883. [DOI] [PubMed] [Google Scholar]
- 9.Kuo SC, Chang SC, Wang HY. et al. Emergence of extensively drug-resistant Acinetobacter baumannii complex over 10 years: nationwide data from the Taiwan Surveillance of Antimicrobial Resistance (TSAR) program. BMC Infect Dis 2012; 12: 200. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Higgins PG, Lehmann M, Wisplinghoff H. et al. gyrB multiplex PCR to differentiate between Acinetobacter calcoaceticus and Acinetobacter genomic species 3. J Clin Microbiol 2010; 48: 4592–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Eleventh Edition: M07. 2019. [Google Scholar]
- 12.Ellington MJ, Kistler J, Livermore DM. et al. Multiplex PCR for rapid detection of genes encoding acquired metallo-β-lactamases. J Antimicrob Chemother 2007; 59: 321–2. [DOI] [PubMed] [Google Scholar]
- 13.Queenan AM, Bush K.. Carbapenemases: the versatile β-lactamases. Clin Microbiol Rev 2007; 20: 440–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Chen FJ, Huang WC, Liao YC. et al. Molecular epidemiology of emerging carbapenem resistance in Acinetobacter nosocomialis and Acinetobacter pittii in Taiwan, 2010 to 2014. Antimicrob Agents Chemother 2019; 63: e02007-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Monstein HJ, Ostholm-Balkhed A, Nilsson MV. et al. Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae. APMIS 2007; 115: 1400–8. [DOI] [PubMed] [Google Scholar]
- 16.Nuesch-Inderbinen MT, Hachler H, Kayser FH.. Detection of genes coding for extended-spectrum SHV β-lactamases in clinical isolates by a molecular genetic method, and comparison with the E test. Eur J Clin Microbiol Infect Dis 1996; 15: 398–402. [DOI] [PubMed] [Google Scholar]
- 17.Woodford N, Fagan EJ, Ellington MJ.. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases. J Antimicrob Chemother 2006; 57: 154–5. [DOI] [PubMed] [Google Scholar]
- 18.Perez-Perez FJ, Hanson ND.. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002; 40: 2153–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.CLSI. Performance Standards for Antimicrobial Susceptibility Testing—Twenty-Ninth Edition: M100. 2019. [Google Scholar]
- 20.Chiu SK, Ma L, Chan MC. et al. Carbapenem nonsusceptible Klebsiella pneumoniae in Taiwan: dissemination and increasing resistance of carbapenemase producers during 2012-2015. Sci Rep 2018; 8: 8468. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Chang YT, Siu LK, Wang JT. et al. Resistance mechanisms and molecular epidemiology of carbapenem-nonsusceptible Escherichia coli in Taiwan, 2012-2015. Infect Drug Resist 2019; 12: 2113–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Petty LA, Henig O, Patel TS. et al. Overview of meropenem-vaborbactam and newer antimicrobial agents for the treatment of carbapenem-resistant Enterobacteriaceae. Infect Drug Resist 2018; 11: 1461–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Livermore DM, Mushtaq S, Warner M. et al. In vitro activity of cefepime/zidebactam (WCK 5222) against Gram-negative bacteria. J Antimicrob Chemother 2017; 72: 1373–85. [DOI] [PubMed] [Google Scholar]
- 24.Moya B, Barcelo IM, Cabot G. et al. In vitro and in vivo activities of β-lactams in combination with the novel β-lactam enhancers zidebactam and WCK 5153 against multidrug-resistant metallo-β-lactamase-producing Klebsiella pneumoniae. Antimicrob Agents Chemother 2019; 63: e00128-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Thomson KS, AbdelGhani S, Snyder JW. et al. Activity of cefepime-zidebactam against multidrug-resistant (MDR) Gram-negative pathogens. Antibiotics (Basel) 2019; 8: 32. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Sfeir MM, Hayden JA, Fauntleroy KA. et al. EDTA-modified carbapenem inactivation method: a phenotypic method for detecting metallo-β-lactamase-producing Enterobacteriaceae. J Clin Microbiol 2019; 57: e01757-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Gallagher JC. Omadacycline: a modernized tetracycline. Clin Infect Dis 2019; 69: S1–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Kishii R, Yamaguchi Y, Takei M.. In vitro activities and spectrum of the novel fluoroquinolone lascufloxacin (KRP-AM1977). Antimicrob Agents Chemother 2017; 61: e00120-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Jorgensen SCJ, Mercuro NJ, Davis SL. et al. Delafloxacin: place in therapy and review of microbiologic, clinical and pharmacologic properties. Infect Dis Ther 2018; 7: 197–217. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Morrissey I, Olesky M, Hawser S. et al. In vitro activity of eravacycline against Gram-negative bacilli isolated in clinical laboratories worldwide from 2013 to 2017. Antimicrob Agents Chemother 2020; 64: e01699-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Jean SS, Lee NY, Tang HJ. et al. Carbapenem-resistant Enterobacteriaceae infections: Taiwan aspects. Front Microbiol 2018; 9: 2888. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Sheng WH, Badal RE, Hsueh PR. et al. Distribution of extended-spectrum β-lactamases, AmpC β-lactamases, and carbapenemases among Enterobacteriaceae isolates causing intra-abdominal infections in the Asia-Pacific region: results of the study for Monitoring Antimicrobial Resistance Trends (SMART). Antimicrob Agents Chemother 2013; 57: 2981–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Sepp E, Andreson R, Balode A. et al. Phenotypic and molecular epidemiology of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli in northern and eastern Europe. Front Microbiol 2019; 10: 2465. [DOI] [PMC free article] [PubMed] [Google Scholar]
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