To the Editor,
Gram-negative organisms cause a broad range of infections such as urinary tract infection, respiratory infections, and bacteraemia. As a group, they are also among the most common cause of healthcare-associated infections, resulting in increased mortality, morbidity, length-of-stay, and healthcare costs.
1
,2
Drug resistance further contributes to increased mortality.3
Enterobacterales as a group cause are the most commonly isolated pathogens, but non-fermenting Gram-negative bacilli (NFGNB) have more intrinsic resistance mechanisms. They can also acquire new resistance mechanisms, which further limits therapeutic options. Pseudomonas aeruginosa, Acinetobacter baumannii complex, Stenotrophomonas maltophilia, and Elizabethkingia anophelis are NFGNB associated with hospital-acquired infections. While S. maltophilia and Elizabethkingia spp. commonly have intrinsic β-lactamase production that result in carbapenem resistance, P. aeruginosa and A. baumannii complex isolates may acquire carbapenem-resistance through various mechanisms including carbapenemase production. The presence of carbapenemases in particular, result in concurrent resistance to various β-lactams.In 2018, a new term ‘difficult-to-treat resistance’ (DTR) was proposed, defined as resistance to all β-lactams and quinolones to reflect resistance to first-line agents for treating Gram-negative infections.
3
In a retrospective cohort study, 2.3% (101/4,493) of P. aeruginosa and 18.3% (183/999) of A. baumannii bloodstream infections were DTR.3
Infections with DTR isolates in this study were shown to have a higher mortality rate even compared to carbapenem-resistant and quinolone-resistant infections.3
As drug resistance becomes more problematic, some of the newer β-lactam-β-lactamase-inhibitor (BLBLI) combinations are now recommended as first line therapy for DTR organisms because of their ability to inhibit carbapenemases.4
,- Tamma P.D.
- Aitken S.L.
- Bonomo
- et al.
Infectious Diseases Society of America guidance on the treatment of extended-spectrum β-lactamase producing Enterobacterales (ESBL-E), carbapenem-resistant Enterobacterales (CRE), and Pseudomonas aeruginosa with difficult-to-treat resistance (DTR-P. aeruginosa).
Clin Infect Dis. 2021; 72: e169-e183
5
These include ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam. The new siderophore antibiotic cefiderocol has also been reported to demonstrate in vitro susceptibility against isolates that produce various carbapenemase, including NDM, and is recommended for management.4
,- Tamma P.D.
- Aitken S.L.
- Bonomo
- et al.
Infectious Diseases Society of America guidance on the treatment of extended-spectrum β-lactamase producing Enterobacterales (ESBL-E), carbapenem-resistant Enterobacterales (CRE), and Pseudomonas aeruginosa with difficult-to-treat resistance (DTR-P. aeruginosa).
Clin Infect Dis. 2021; 72: e169-e183
5
These newer generation BLBLI have been suggested as possible treatment options for drug resistant Gram-negative organisms. In addition, other drug classes have also seen new members such as omadacycline and eravacycline (fluorocyclines), plazomicin (aminoglycoside), and delafloxacin (fluoroquinolone).We have previously demonstrated limited in vitro susceptibility of ceftazidime-avibactam and ceftolozane-tazobactam in DTR P. aeruginosa isolates in our setting.
6
However, the correlation between these phenotypes and resistance mechanisms (carbapenemases in particular) was not performed. To further assess the feasibility of new antibiotics including BLBLI combinations as treatment options for non-fermenters, susceptibility testing was performed on a collection of NFGNB. Testing for carbapenemase genes was also performed on DTR P. aeruginosa and A. baumannii complex isolates to identify the presence of common carbapenemase in these isolates. These genotypic results were then compared against phenotypic susceptibility of BLBLI combinations and cefiderocol. Concurrent testing to new non-β-lactam antibiotics was also performed to identify alternative treatment options.A collection of drug resistant Gram-negative organisms were tested by broth microdilution using Sensititre MDRGN2F plates (ThermoFisher, Singapore). The included isolates were P. aeruginosa, A. baumannii complex, S. maltophilia, and E. anophelis. All isolates were identified by MALDI-TOF (MALDI Biotyper; Bruker, USA). Duplicate isolates of the same bacterial species from the same patient were excluded.
Pseudomonas aeruginosa and A. baumannii complex were selected as they are the two most common NFGNB associated with nosocomial infections.
1
DTR P. aeruginosa and DTR A. baumannii complex isolates from all clinical samples in 2021 were included. DTR in P. aeruginosa and A. baumannii complex was defined as resistance against ceftazidime, cefepime, piperacillin-tazobactam, imipenem, meropenem, and ciprofloxacin which was routinely tested using Vitek 2 (bioMérieux, France) based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. For A. baumannii complex isolates, EUCAST non-species breakpoints were used where species-specific breakpoints were unavailable. Three historical NDM-positive A. baumannii complex isolates were also included.Stenotrophomonas maltophilia and E. anophelis were included as they are causes of nosocomial infections in our local setting, and also have intrinsic resistance to carbapenems. These isolates were not pre-selected based on routine antimicrobial susceptibility profiles. Stenotrophomonas maltophilia and E. anophelis isolates from blood cultures between 2019–2022 were included.
Susceptibility testing by the Sensititre MDRGN2F plates was performed as per manufacturer instructions. The MDRGN2F plates included the following antibiotics [tested minimum inhibitory concentration (MIC) range]: meropenem-vaborbactam (0.008–16 mg/L; vaborbactam fixed concentration 8 mg/L); omadacycline (0.12–8 mg/L); plazomicin (0.12–4 mg/L); imipenem-relebactam (0.03–16 mg/L; relebactam fixed concentration 4 mg/L); ceftazidime-avibactam (0.5–32 mg/L; avibactam fixed concentration 4 mg/L); eravacycline (0.03–8 mg/L); delafloxacin (0.12–1 mg/L); cefiderocol (0.03–32 mg/L); ceftolozane-tazobactam (0.06–8 mg/L; tazobactam fixed concentration 4 mg/L); levofloxacin (0.25–4 mg/L); meropenem (0.25–8 mg/L); imipenem (2–16 mg/L); amikacin (16–32 mg/L). Levofloxacin, meropenem, and imipenem were not analysed as all were resistant because of the selection criteria for P. aeruginosa and A. baumannii complex isolates. Amikacin was not analysed because of the narrow tested MIC range. Quality control was performed using Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 with all antimicrobial results being within the expected range.
EUCAST breakpoints are available for P. aeruginosa for meropenem-vaborbactam, imipenem-relebactam, ceftazidime-avibactam, ceftolozane-tazobactam, and cefiderocol. PK-PD (non-species-related) breakpoints are also available for these antibiotics. The P. aeruginosa and non-species-related breakpoints are currently the same (meropenem-vaborbactam S≤8, R>8; imipenem-relebactam S≤2, R>2; ceftazidime-avibactam S≤8, R>8; ceftolozane-tazobactam S≤4, R>4; cefiderocol S≤2, R>2). Thus, these same breakpoints were applied for all bacterial species in this study. There are currently no interpretive breakpoints available for omadacycline, eravacycline, delafloxacin, or plazomicin.
Pseudomonas aeruginosa and A. baumannii complex isolates were also screened for carbapenemase carriage via polymerase chain reaction (PCR). The following carbapenemase genes were targeted: blaKPC, blaGES, blaOXA-58, blaOXA-48, blaOXA-40, blaOXA-23, blaNDM, blaVIM, and blaIMP as previously described.
7
NDM, KPC, and OXA carbapenemases are the most commonly identified carbapenemases in carbapenemase-producing Enterobacterales locally,8
while GES, VIM, and IMP are other commonly described carbapenemases in P. aeruginosa.9
A total of 34 DTR P. aeruginosa, 28 DTR A. baumannii complex, 15 S. maltophilia, and 7 E. anophelis isolates were included. The P. aeruginosa isolates consisted of three blood, seven respiratory, 13 urine, and 11 miscellaneous (tissue, wound, or fluid) isolates. The A. baumannii complex isolates consisted of five blood, four respiratory, 11 urine, and eight miscellaneous isolates. All S. maltophilia and E. anophelis were blood isolates.
MIC50 and MIC90 of all antibiotics for all isolates are shown in Table 1. Susceptibility and resistance rates are also shown for meropenem-vaborbactam, imipenem-relebactam, ceftazidime-avibactam, ceftolozane-tazobactam, and cefiderocol for all isolates. Due to the lack of breakpoints including PK-PD (non-species-related) breakpoints, susceptibility/resistance rates could not be reported for omadacycline, eravacycline, delafloxacin, and plazomicin. The MIC distributions of these antibiotics are shown in Table 2.
Table 1Susceptibility rates, MIC50, and MIC90 of newer generation β-lactam-β-lactamase-inhibitor combinations and cefiderocol
| Antibiotic | S | MIC50 | MIC90 |
|---|---|---|---|
| Pseudomonas aeruginosa | |||
| Meropenem-vaborbactam | 8.8% (3/34) | >16 | >16 |
| Imipenem-relebactam | 5.9% (2/34) | >16 | >16 |
| Ceftazidime-avibactam | 5.9% (2/34) | >32 | >32 |
| Ceftolozane-tazobactam | 14.7% (5/34) | >8 | >8 |
| Cefiderocol | 52.9% (18/34) | 2 | 8 |
| Acinetobacter baumannii complex | |||
| Meropenem-vaborbactam | 0.0% (0/28) | >16 | >16 |
| Imipenem-relebactam | 0.0% (0/28) | >16 | >16 |
| Ceftazidime-avibactam | 3.6% (1/28) | >32 | >32 |
| Ceftolozane-tazobactam | 3.6% (1/28) | >8 | >8 |
| Cefiderocol | 60.7% (17/28) | 1 | 8 |
| Stenotrophomonas maltophilia | |||
| Meropenem-vaborbactam | 0.0% (0/15) | >16 | >16 |
| Imipenem-relebactam | 0.0% (0/15) | >16 | >16 |
| Ceftazidime-avibactam | 60.0% (9/15) | 8 | >32 |
| Ceftolozane-tazobactam | 40.0% (6/15) | >8 | >8 |
| Cefiderocol | 93.3% (14/15) | 1 | 8 |
| Elizabethkingia anophelis | |||
| Meropenem-vaborbactam | 0.0% (0/7) | >16 | >16 |
| Imipenem-relebactam | 0.0% (0/7) | >16 | >16 |
| Ceftazidime-avibactam | 0.0% (0/7) | 32 | 32 |
| Ceftolozane-tazobactam | 0.0% (0/7) | >8 | >8 |
| Cefiderocol | 14.3% (1/7) | 32 | >32 |
S, susceptible, standard dosing regimen; MIC50, MIC at which at least 50% of isolates were inhibited; MIC90, MIC at which at least 90% of isolates were inhibited.
Table 2MIC distribution, MIC50, and MIC90 to omadacycline, eravacycline, delafloxacin, and plazomicin
| Antibiotic | Minimum inhibitory concentration (MIC), mg/L | MIC50 | MIC90 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.03 | 0.06 | 0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | >8 | |||
| Pseudomonas aeruginosa | ||||||||||||
| Omadacycline | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 34 | >8 | >8 | ||
| Eravacycline | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 14 | 20 | >8 | >8 |
| Delafloxacin | 0 | 0 | 0 | 4 | 30 | >1 | >1 | |||||
| Plazomicin | 0 | 0 | 0 | 1 | 2 | 12 | 19 | >4 | >4 | |||
| Acinetobacter baumannii complex | ||||||||||||
| Omadacycline | 1 | 2 | 1 | 3 | 11 | 7 | 0 | 3 | 2 | >8 | ||
| Eravacycline | 1 | 2 | 0 | 5 | 14 | 3 | 1 | 1 | 0 | 1 | 0.5 | 2 |
| Delafloxacin | 1 | 0 | 1 | 3 | 23 | >1 | >1 | |||||
| Plazomicin | 0 | 0 | 1 | 2 | 3 | 1 | 21 | >4 | >4 | |||
| Stenotrophomonas maltophilia | ||||||||||||
| Omadacycline | 0 | 0 | 0 | 1 | 5 | 5 | 1 | 3 | 4 | >8 | ||
| Eravacycline | 0 | 0 | 1 | 0 | 7 | 4 | 1 | 0 | 2 | 0.5 | 8 | |
| Delafloxacin | 2 | 0 | 4 | 3 | 6 | 1 | >1 | |||||
| Plazomicin | 0 | 0 | 0 | 0 | 0 | 1 | 14 | >4 | >4 | |||
| Elizabethkingia anophelis | ||||||||||||
| Omadacycline | 0 | 0 | 0 | 0 | 1 | 3 | 2 | 1 | 4 | >8 | ||
| Eravacycline | 0 | 0 | 0 | 0 | 0 | 3 | 4 | 0 | 0 | 2 | 2 | |
| Delafloxacin | 3 | 3 | 1 | 0 | 0.25 | 0.5 | ||||||
| Plazomicin | 0 | 0 | 0 | 0 | 0 | 0 | 7 | >4 | >4 | |||
a Indicates isolates with MIC outside of tested range.
Of the 34 DTR P. aeruginosa, 18 isolates (52.9%) were positive for carbapenemase genes. Eight were NDM-positive, and ten were IMP-1-positive. Sixteen isolates were negative for carbapenemase genes. All carbapenemase-positive P. aeruginosa were resistant to all BLBLI combinations. The susceptibility rates of carbapenemase-negative P. aeruginosa are as follows: meropenem-vaborbactam (18.8%, 3/16); imipenem-relebactam (12.5%, 2/16); ceftazidime-avibactam (18.8%, 3/16); ceftolozane-tazobactam (31.3%, 5/16). None of the NDM-positive isolates were susceptible to cefiderocol. Fifty percent (5/10) of IMP-positive isolates and 81.3% (13/16) of carbapenemase-negative isolates were susceptible to cefiderocol.
Of the 28 DTR A. baumannii complex, seven isolates (25.0%) were carbapenemase-positive. One was IMP-positive, three were OXA-23-positive, one was OXA-58 and NDM-positive, and two were OXA-23 and NDM-positive. All carbapenemase-positive isolates were resistant to all BLBLI combinations. However, of the carbapenemase-negative isolates, only one isolate was susceptible to both ceftazidime-avibactam and ceftolozane-tazobactam (4.8%, 1/21). All isolates with NDM were resistant to cefiderocol. The single IMP-1-positive isolate was susceptible to cefiderocol, while two of three OXA-23-positive isolates were susceptible to cefiderocol (66.7%). Of the 21 carbapenemase-negative isolates, 66.7% were susceptible to cefiderocol (14/21).
Phenotypic susceptibility to the tested BLBLI and cefiderocol stratified by carbapenemase genotype is presented in Table 3.
Table 3Susceptibility rates of DTR P. aeruginosa and A. baumannii complex isolates to newer generation β-lactam-β-lactamase-inhibitor combinations and cefiderocol, stratified by carbapenemase
| Antibiotic | |||||
|---|---|---|---|---|---|
| Meropenem-vaborbactam | Imipenem-relebactam | Ceftazidime-avibactam | Ceftolozane-tazobactam | Cefiderocol | |
| Pseudomonas aeruginosa (n=34) | |||||
| Carbapenemase-negative (n=16) | 18.8% (3/16) | 12.5% (2/16) | 18.8% (3/16) | 31.3% (5/16) | 81.3% (13/16) |
| IMP (n=10) | 0.0% (0/10) | 0.0% (0/10) | 0.0% (0/10) | 0.0% (0/10) | 50% (5/10) |
| NDM (n=8) | 0.0% (0/8) | 0.0% (0/8) | 0.0% (0/8) | 0.0% (0/8) | 0.0% (0/8) |
| Acinetobacter baumannii complex (n=28) | |||||
| Carbapenemase-negative (n=21) | 0.0% (0/21) | 0.0% (0/21) | 4.8% (1/21) | 4.8% (1/21) | 66.7% (14/21) |
| IMP (n=1) | 0.0% (0/1) | 0.0% (0/1) | 0.0% (0/1) | 0.0% (0/1) | 100.0% (1/1) |
| OXA-23 (n=3) | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 0.0% (0/3) | 66.7% (2/3) |
| OXA-58 and NDM (n=1) | 0.0% (0/1) | 0.0% (0/1) | 0.0% (0/1) | 0.0% (0/1) | 0.0% (0/1) |
| OXA-23 and NDM (n=2) | 0.0% (0/2) | 0.0% (0/2) | 0.0% (0/2) | 0.0% (0/2) | 0.0% (0/2) |
Treatment options for drug-resistant Gram-negative bacteria remains limited despite availability of newer generation antimicrobials. We demonstrate that newer-generation BLBLI combinations had limited in vitro activity against DTR P. aeruginosa, DTR A. baumannii complex, S. maltophilia, and E. anophelis. Cefiderocol also had poor activity against E. anophelis but 52.9%, 60.7%, and 93.3% of DTR P. aeruginosa, DTR A. baumannii complex, and S. maltophilia demonstrated in vitro susceptibility, respectively. High rates of carbapenemase were identified among our DTR P. aeruginosa isolates (>52.9%), while only 25% of DTR A. baumannii complex isolates were carbapenemase positive. Isolates which were carbapenemase-positive were more likely to be resistant to BLBLI combinations, and all NDM-positive isolates were resistant to cefiderocol. The association of IMP-positive isolates with cefiderocol resistance was weaker. While susceptibility rates of BLBLI combinations were higher in carbapenemase-negative P. aeruginosa, overall activity remains limited, indicating likely presence of other non-carbapenemase mediated resistance mechanisms.
Currently, screening of drug-resistant P. aeruginosa and A. baumannii complex isolates for the presence of carbapenemase (genes) is not routinely performed in our local setting. Our data indicate that the presence of carbapenemase genes is predictive of resistance to BLBLI combinations (particularly for P. aeruginosa), and in the case of NDM, resistance to cefiderocol too. High rates of resistance to BLBLI were seen in A. baumannii complex isolates, regardless of the presence of carbapenemase. In our context, IMP and NDM [both metallo-β-lactamases (MBL)] predominated in carbapenemase-positive P. aeruginosa, but none of the P. aeruginosa isolates in our collection was positive for group A (such as KPC or GES) or group D (OXA-group) carbapenemases. This is consistent with global trends, where group D carbapenemases are rarely described, with higher rates of group A carbapenemases, followed by MBLs.
9
None of these MBL-positive P. aeruginosa isolates were susceptible to the new BLBLI combinations which is expected as the β-lactamase-inhibitors (vaborbactam, avibactam, relebactam, and tazobactam) do not inhibit MBL. Carbapenemase testing in P. aeruginosa can predict resistance to BLBLI when positive for a MBL. However, phenotypic testing is still required confirming susceptibility in carbapenemase-negative isolates. In regions where group A carbapenemases are more commonly seen in P. aeruginosa, phenotypic susceptibility to other newer BLBLI may still be retained. A few A. baumannii complex isolates with MBLs were identified although globally, group D Carbapenemases (OXA) are the most common carbapenemases identified in A. baumannii, including intrinsic OXA-51 genes.Further typing of carbapenemase-positive isolates was not performed in this study. However, it is important to note that >50% of DTR P. aeruginosa and 25% of DTR A. baumannii complex isolates were positive for carbapenemase. Clonal transmission of NDM-positive P. aeruginosa was demonstrated previously.
10
Of note, IMP and VIM were identified in both P. aeruginosa and A. baumannii complex isolates. Clonal transmission of bacterial isolates or horizontal transmission of mobile genetic elements is another aspect with potential infection control implications with regards to carbapenemase testing in NFGNB. This may be important to consider in future studies as part of international efforts to combat drug resistance in healthcare settings.The MICs of P. aeruginosa for omadacycline, eravacycline, delafloxacin, and plazomicin were generally on the higher end of the tested range indicating limited in vitro activity. MICs to omadacycline and eravacycline may be due to intrinsic class resistance of P. aeruginosa to tetracyclines. In addition, high rates of acquired resistance (to aminoglycosides and fluoroquinolones) is expected as these isolates were selected on basis of having DTR phenotype. Similarly, MICs of delafloxacin and plazomicin for A. baumannii complex isolates were also high. The MICs to omadacycline and eravacycline were more variable, suggesting possible in vitro activity amongst DTR isolates. The MIC50 and MIC90 for eravacycline were similar to those reported in other A. baumannii isolates,
11
but higher for omadacycline.12
Stenotrophomonas maltophilia and E. anophelis are known to have intrinsic resistance mechanisms against aminoglycosides which reflects limited inhibition by plazomicin. Variable MICs were seen for omadacycline, eravacycline, and delafloxacin. Further data are required to establish breakpoints with which to interpret whether these antibiotics are suitable treatment options for these organisms. Cefiderocol may be a possible treatment option of S. maltophilia. Ceftazidime-avibactam and ceftolozane-tazobactam also has better activity for S. maltophilia compared to other BLBLI, although clinical data are required given the presence of intrinsic β-lactamases. All BLBLI and cefiderocol appear to have limited activity against E. anophelis.
With the exception of >90% susceptibility to cefiderocol seen in S. maltophilia, our data indicate that newer generation antibiotics do not appear to offer significant advantage for the tested isolates. In DTR P. aeruginosa and A. baumannii complex isolates, the activity of BLBLI is limited. While some of this is correlated with the presence of carbapenemase genes, particularly P. aeruginosa, high rates of resistance are seen even in carbapenemase-negative isolates. Similarly, almost all A. baumannii complex isolates were resistant to BLBLI combinations. The fluorocyclines may have activity for A. baumannii complex, S. maltophilia, and E. anophelis infections, although additional clinical data are required.
Acknowledgements
We would like to thank ThermoFisher Scientific (Singapore) Pte Ltd for providing MDRGN2F plates that were used in this project. ThermoFisher was not involved in study design, data collection and analysis, or decision to publish.
Conflicts of interest and sources of funding
The authors state that there are no conflicts of interest to disclose.
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Article info
Publication history
Published online: December 23, 2022
Publication stage
In Press Corrected ProofIdentification
Copyright
© 2022 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.
