Multidrug-resistant (MDR) Gram-negative organisms are a major health concern due to lack of effective therapy. Emergence of resistance to newer agents like ceftazidime-avibactam (CZA) further magnifies the problem. In this context, combination therapy of CZA with other antimicrobials may have potential in treating these pathogens. Unfortunately, there are limited data regarding these combinations.
KEYWORDS: Klebsiella pneumoniae, Pseudomonas aeruginosa, antibiotic combinations, ceftazidime-avibactam
ABSTRACT
Multidrug-resistant (MDR) Gram-negative organisms are a major health concern due to lack of effective therapy. Emergence of resistance to newer agents like ceftazidime-avibactam (CZA) further magnifies the problem. In this context, combination therapy of CZA with other antimicrobials may have potential in treating these pathogens. Unfortunately, there are limited data regarding these combinations. Therefore, the objective of this study was to evaluate CZA in combination with amikacin (AMK), aztreonam (AZT), colistin (COL), fosfomycin (FOS), and meropenem (MEM) against 21 carbapenem-resistant Klebsiella pneumoniae and 21 MDR Pseudomonas aeruginosa strains. The potential for synergy was evaluated via MIC combination evaluation and time-kill assays. All strains were further characterized by whole-genome sequencing, quantitative real-time PCR, and SDS-PAGE analysis to determine potential mechanisms of resistance. Compared to CZA alone, we observed a 4-fold decrease in CZA MICs for a majority of K. pneumoniae strains and at least a 2-fold decrease for most P. aeruginosa isolates in the majority of combinations tested. In both P. aeruginosa and K. pneumoniae strains, CZA in combination with AMK or AZT was synergistic (≥2.15-log10 CFU/ml decrease). CZA-MEM was effective against P. aeruginosa and CZA-FOS was effective against K. pneumoniae. Time-kill analysis also revealed that the synergy of CZA with MEM or AZT may be due to the previously reported restoration of MEM or AZT activity against these organisms. Our findings show that CZA in combination with these antibiotics has potential for therapeutic options in difficult to treat pathogens. Further evaluation of these combinations is warranted.
INTRODUCTION
Gram-negative resistance is a growing concern in health care due to lack of viable antimicrobial therapeutic alternatives. Carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae, and multidrug-resistant (MDR) Pseudomonas aeruginosa are considered to be urgent public health threats by the CDC, requiring urgent and aggressive actions to contain their dissemination and find new therapies for these organisms (1).
Klebsiella spp. are the highest percentage of health care-associated CRE (11%), and the estimated number of deaths attributed to these infections was 520 out of 7,900 infected patients in 2012 (1). According to a review done in 2017 assessing the evolution of CRE, Klebsiella pneumoniae producing carbapenemase (KPC) was the most common transmissible CRE worldwide (2). Another global 2017 systemic review and meta-analysis evaluating the mortality of patients infected by KPC producers revealed that the collective mortality was 42.14% among 2,462 patients infected with KPC producers versus 21.16% in those infected with carbapenem-susceptible K. pneumoniae (3). Furthermore, compared to non-ESBL-producing Enterobacteriaceae, including Klebsiella pneumoniae, patients infected with these pathogens have increased mortality primarily due to a delay in the administration of active therapy (4, 5).
As stated above, in addition to CRE, MDR P. aeruginosa is considered a serious public health care threat by the Centers for Disease Control and Prevention. It is estimated that there are 51,000 health care-associated Pseudomonas aeruginosa infections in the United States per year, with 13% of these classified as multidrug resistant, contributing to 400 deaths annually (1). Some strains of P. aeruginosa have been reported to be resistant to almost all antimicrobials, including aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems, with prior antibiotic exposure as one of the major contributing risk factors (6).
Ceftazidime-avibactam (CZA) is a relatively new combination of a third-generation cephalosporin and a novel β-lactamase inhibitor. Avibactam (AVI) reestablishes the in vitro activity of ceftazidime against Ambler class A, class C, and some class D β-lactamase-producing pathogens (7, 8). CZA was shown to have a clinical efficacy similar to that of meropenem (MEM) and doripenem for the treatment of complicated infections (9–12). It has inhibitory activity against β-lactamase-producing Gram-negative pathogens, ESBLs, AmpC β-lactamases, and KPC-producing K. pneumoniae and MDR P. aeruginosa (7, 8, 13–15). This β-lactam–β-lactamase combination is currently FDA approved for the treatment of complicated intra-abdominal infections used in combination with metronidazole, complicated urinary tract infections, and hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia (16–18).
Although the spectrum of activity of CZA is vast, there are reports of emergence of resistance to this therapy. A recent study demonstrates that a single substitution mutation of the KPC-2 gene will disable the inhibitory effects of avibactam against KPC against β-lactamases (19). This suggests that the emergence of resistance may threaten the efficacy of the CZA in the near future. Similarly, there are reports of mutations that result in variant KPC-3 enzymes that significantly reduce CZA susceptibility and generally behave like ESBLs (20). With the increased use of CZA, it is expected that resistance will continue to develop and plasmids carrying mutant genes may spread by horizontal gene transfer. (21). Winkler and colleagues discovered that clinical P. aeruginosa isolates resistant to CZA were likely due to diminished outer membrane permeability and/or overexpressed efflux pumps (22). Other studies have shown P. aeruginosa to have the ability to regrow in the presence of CZA or become resistant by the overexpression of AmpC (23, 24).
Combination therapy has been proven to be useful against various resistant organisms to achieve maximum antimicrobial effect. There is little information available for CZA combination with other antimicrobial agents against these organisms, and the objective of this study was to compare CZA alone and in combination with standard and novel antimicrobials against MDR strains of K. pneumoniae and P. aeruginosa by using tools like combination MIC testing and time-kill assays.
RESULTS
Susceptibility testing.
The MIC values of CZA alone and in combination with the adjunctive antimicrobials against K. pneumoniae and P. aeruginosa are listed in Table 1. Acquired genes encoding resistance to aminoglycosides, β-lactams, quinolones, fosfomycin (FOS), and colistin (COL), among others, are listed in Table 2. β-Lactam resistance mechanisms among the Pseudomonas aeruginosa isolates, including the isolates used for time-kill assays, are listed in Table 3.
TABLE 1.
MIC of CZA alone and in combination with adjunctive antimicrobials against K. pneumoniae and P. aeruginosa
| Organism | MIC (μg/ml)a
|
|||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CZA | CZA-AMK | CZA-AZTb | CZA-COL | MEM | CZA-MEMb | FOS | CZA-FOSb | AMK | AZT | AZT-AVI | COL | MEM-AVI | FOS-AVI | |
| K. pneumoniae (n = 21) | ||||||||||||||
| R8375 | 2 | 0.25 | 0.25 | 0.5 | 32 | 0.25 | 32 | 0.5 | 16 | >256 | 0.5 | 0.5 | 0.03 | 16 |
| R9009 | 2 | 0.25 | 0.25 | 1 | 32 | <0.13 | 64 | 0.25 | 16 | >256 | 1 | 0.5 | 0.02 | 16 |
| R9011 | 0.5 | 0.25 | 0.25 | 0.5 | 32 | 0.25 | 128 | 0.5 | 1 | >256 | 0.25 | 1 | 0.03 | 16 |
| R9064 | 1 | <0.13 | 0.25 | 1 | 32 | 0.25 | 256 | 0.5 | 2 | >256 | 0.25 | 0.5 | 0.06 | 64 |
| R9158 | 1 | 1 | 0.25 | 1 | 64 | 0.25 | 128 | 0.25 | 32 | 16 | 0.25 | 2 | 0.25 | 64 |
| R10197 | 0.5 | <0.13 | 0.25 | 0.5 | 32 | <0.13 | 32 | 0.13 | 2 | >256 | 0.25 | 1 | 0.06 | 32 |
| R10276 | 0.5 | 0.25 | 0.5 | 0.5 | 32 | <0.13 | 128 | 0.25 | 32 | >256 | 0.5 | 0.25 | 0.03 | 128 |
| R10277 | 0.5 | 0.25 | 0.25 | 0.25 | 64 | 0.5 | 64 | 0.25 | 32 | >256 | 0.5 | 1 | 0.02 | 64 |
| R10278 | 0.5 | <0.13 | 0.25 | 0.5 | 32 | <0.13 | 32 | 0.5 | 32 | >256 | 0.25 | 0.5 | 0.02 | 32 |
| R10428 | 1 | 0.13 | 0.25 | 0.5 | 32 | <0.13 | 32 | 0.5 | 2 | >256 | 0.25 | 0.5 | 0.02 | 32 |
| R10499 | 0.5 | <0.13 | <0.13 | 0.5 | 32 | <0.13 | 128 | 0.13 | 2 | >256 | 0.13 | 0.5 | 0.02 | 64 |
| R10500 | 0.5 | 0.13 | <0.13 | <0.13 | 32 | 0.25 | 64 | 0.25 | 2 | >256 | 0.25 | 1 | 0.02 | 32 |
| R10501 | 1 | 0.25 | 0.5 | 1 | >64 | 0.25 | 128 | <0.13 | 32 | >256 | 0.5 | 1 | 0.13 | 128 |
| R10502 | 0.5 | <0.13 | <0.13 | 0.5 | 16 | 0.5 | 32 | <0.13 | 2 | >256 | 0.25 | 1 | 0.01 | 32 |
| R10503 | 0.5 | 0.13 | <0.13 | 0.5 | 32 | 0.5 | 64 | 0.13 | 32 | >256 | 0.25 | 0.5 | 0.01 | 64 |
| R10504 | 1 | <0.06 | <0.13 | 1 | >64 | 0.5 | 256 | 0.25 | 64 | >256 | 0.25 | 0.5 | 0.13 | 128 |
| R10505 | 0.5 | <0.06 | <0.13 | 0.5 | 16 | 0.25 | 128 | 0.25 | 32 | >256 | 0.25 | 1 | 0.00 | 16 |
| R10506 | 0.5 | <0.13 | <0.13 | 0.5 | 32 | <0.13 | 64 | 0.25 | 4 | >256 | 0.13 | 1 | 0.02 | 16 |
| R10507 | 0.5 | <0.06 | 0.25 | 0.25 | 16 | <0.13 | 32 | 0.25 | 32 | >256 | 0.13 | 1 | 0.02 | 8 |
| R10508 | 0.5 | <0.13 | <0.13 | 0.13 | 16 | <0.13 | >512 | 0.25 | 2 | >256 | 0.13 | 1 | 0.13 | >512 |
| R10831 | 4 | <0.13 | 1 | 4 | >64 | 4 | 256 | 1 | 2 | >256 | 0.5 | 0.5 | 0.25 | 256 |
| P. aeruginosa (n = 21) | ||||||||||||||
| R8381 | 2 | 2 | 2 | 2 | 4 | 0.5 | >512 | 1 | 2 | 2 | 1 | 1 | 2 | >512 |
| R9010 | 8 | 2 | 4 | 8 | 16 | 8 | >512 | 4 | 32 | 16 | 16 | 2 | 4 | 256 |
| R9042 | 2 | 0.25 | 2 | 0.5 | 16 | 1 | 64 | 2 | 8 | >256 | 16 | 0.5 | 16 | 64 |
| R9260 | 8 | 1 | 2 | 8 | 16 | 2 | 256 | 2 | 4 | 8 | 16 | 1 | 8 | 64 |
| R9275 | 64 | 1 | 2 | 4 | 4 | 2 | 4 | 1 | 32 | 32 | 16 | 1 | 0.25 | 2 |
| R9316 | 8 | 4 | 2 | 8 | 32 | 4 | 64 | 8 | 1 | 64 | 64 | 2 | 16 | 32 |
| R9333 | 1 | 0.5 | 1 | 2 | 16 | 1 | 128 | 1 | 4 | 32 | 8 | 1 | 4 | 32 |
| R9647 | 1 | 0.5 | 0.5 | 1 | 1 | 0.25 | 128 | 0.5 | 8 | 16 | 2 | 1 | 0.5 | 64 |
| R10149 | 128 | 64 | 64 | 64 | >64 | 128 | >512 | 128 | 4 | 64 | 64 | 2 | >64 | >512 |
| R10155 | 128 | >64 | 64 | >64 | >64 | 128 | >512 | 64 | >64 | 64 | 16 | 4 | >64 | >512 |
| R10266 | 16 | 8 | 8 | 16 | 32 | 8 | 64 | 16 | >64 | 64 | 64 | 1 | 16 | 64 |
| R10267 | 64 | 16 | 32 | 32 | >64 | 1 | 128 | 16 | 4 | >256 | 256 | 1 | 64 | 64 |
| R10268 | 4 | 2 | 2 | 4 | 16 | 2 | 128 | 1 | >64 | 16 | 4 | 1 | 8 | 32 |
| R10269 | 1 | 0.5 | 1 | 2 | 4 | 1 | >512 | 1 | 8 | 8 | 4 | 1 | 2 | >512 |
| R10270 | 2 | 0.5 | 0.25 | 2 | 8 | 0.5 | >512 | 0.5 | 8 | 64 | 8 | 2 | 4 | 256 |
| R10271 | 1 | 0.5 | 1 | 1 | 32 | 1 | 8 | 0.25 | 2 | >256 | 4 | 1 | 2 | 4 |
| R10272 | 8 | 8 | 8 | 8 | 8 | 8 | 128 | 4 | >64 | 128 | 8 | 1 | 8 | 128 |
| R10273 | 4 | 2 | 4 | 4 | 16 | 4 | >512 | 4 | 8 | 128 | 4 | 1 | 8 | >152 |
| R10274 | 1 | 0.5 | 1 | 1 | 8 | 1 | 512 | 0.25 | 8 | 32 | 4 | 2 | 2 | 512 |
| R10275 | 2 | 0.5 | 1 | 2 | 16 | 2 | 128 | 2 | 4 | 16 | 8 | 2 | 4 | 128 |
| R10378 | 2 | 0.5 | 1 | 2 | 8 | 2 | 256 | 1 | 16 | 16 | 16 | 1 | 8 | 128 |
CZA, ceftazidime-avibactam; AMK, amikacin; AZT, aztreonam; COL, colistin; MEM, meropenem; FOS, fosfomycin.
For the single agents, MICs used in combination with ceftazidime plus AVI, 0.5× MIC of the combination of the single agent + AVI was used. For example, for K. pneumoniae R8375, the combination of CZA-MEM was performed with CZA in the presence of 0.5× MIC of the combination of MEM-AVI (0.015 μg/ml). This was done because CZA plus 0.5× MIC of MEM (16 μg/ml) produced no growth for the entire column.
TABLE 2.
Acquired resistance genes detected among K. pneumoniae and P. aeruginosa
| Organism | CZA MIC (μg/ml) | Genes for aminoglycoside-modifying enzymes | Macrolide resistance | Fosfomycin resistance | Acquired β-lactamase gene(s) |
|---|---|---|---|---|---|
| K. pneumoniae | |||||
| R8375 | 2 | aph(4)-Ia, aph(3′)-Ia, aadA2, aadA1, aac(6′)-Ib, aac(3)-IV | mph(A) | fosA-like | blaKPC-3, blaSHV-12, blaTEM-1, blaSHV-11, blaOXA-2, blaOXA-9 |
| R9009 | 2 | ant(3′)-Ia, aadA2, aac(6′)-Ib | mph(A) | fosA-like | blaKPC-3, blaSHV-12, blaLAP-1, blaTEM-1, blaSHV-11, blaOXA-2, blaOXA-9 |
| R9064 | 1 | ant(3′)-Ia, aac(6′)-Ib | fosA-like | blaKPC-3, blaTEM-1, blaSHV-11, blaOXA-2, blaOXA-9 | |
| R10277 | 0.5 | ant(3′)-Ia, aadA2, aac(6′)-Ib | mph(A) | fosA-like | blaKPC-2, blaTEM-1, blaSHV-11 |
| R10278 | 0.5 | aph(4)-Ia, aph(3′)-Ia, ant(3′)-Ia, aadA2, aac(6′)-Ib, aac(3)-IV | mph(A) | fosA-like | blaKPC-2, blaTEM-1, blaSHV-11 |
| R10501 | 1 | aadA2, aac(6′)-Ib | mph(A) | fosA-like | blaKPC-2 |
| R10506 | 0.5 | aph(3′)-Ia, aadA2, ant(2″)-Ia, aac(6′)-Ib-cr | msr(E), mph(E), mph(A) | fosA-like | blaCTX-M-15, blaOXA-1 |
| R10508 | 0.5 | fosA-like | blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-11 | ||
| R9011 | 0.5 | ant(3′)-Ia, aadA2 | mph(A) | fosA-like | blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-11 |
| R9158 | 1 | ant(3′)-Ia, aadA2 | mph(A) | fosA-like | blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-11 |
| R10197 | 0.5 | ant(3′)-Ia, aadA2 | mph(A) | fosA-like | blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-11 |
| R10279 | aph(4)-Ia, aph(3′)-Ia, ant(3′)-Ia, aadA2, aac(6′)-Ib, aac(3)-IV-like | mph(A) | fosA-like | blaKPC-2, blaTEM-1, blaSHV-11 | |
| R10428 | 1 | aph(6)-Ia, aph(6)-Id | fosA | blaKPC-2, blaCTX-M-15, blaTEM-206, blaSHV-28 | |
| R10499 | 0.5 | ant(3′)-Ia, aadA2 | mph(A) | fosA-like | blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-11 |
| R10500 | 0.5 | ant(3′)-Ia, aadA2 | mph(A) | fosA-like | blaKPC-2, blaCTX-M-15, blaTEM-1, blaSHV-11 |
| R10502 | 0.5 | aphA16, ant(3′)-Ia, ant(2″)-Ia, aac(6′)-Ib | fosA-like | blaKPC-3, blaSHV-12, blaTEM-1, blaSHV-11, blaOXA-2, blaOXA-9 | |
| R10504 | 1 | aph(4)-Ia, aadA2, aadA1, aac(6′)-Ib, aac(3)-IV | fosA-like | blaKPC-3, blaSHV-12, blaTEM-1, blaSHV-11, blaOXA-2, blaOXA-9 | |
| R10507 | 0.5 | ant(3′)-Ia, aadA2, aac(6′)-Ib | mph(A) | fosA-like | blaKPC-3, blaSHV-12, blaTEM-1, blaSHV-11, blaOXA-2, blaOXA-9 |
| R10503 | 0.5 | ant(3′)-Ia, aadA2, aac(6′)-Ib | mph(A) | fosA-like | blaKPC-3, blaSHV-12, blaTEM-1, blaSHV-11, blaOXA-2, blaOXA-9 |
| R10505 | 0.5 | ant(3′)-Ia, aadA2, aac(6′)-Ib | mph(A) | fosA-like | blaKPC-3, blaCTX-M-3, blaSHV-12, blaTEM-1, blaSHV-11, blaOXA-2, blaOXA-9 |
| 10831#1 | 4 | aadA16, aac(6′)-Ib-cr | fosA-like | blaKPC-2, blaTEM-1, blaSHV-11 | |
| P. aeruginosa | |||||
| R9316 | 8 | aph(3′)-IIb-like | fosA-like | ||
| R9647 | 1 | aph(3′)-IIb | fosA | ||
| R10149 | 128 | aph(3′)-Iib, ant(2″)-Ia, aac(6′)-Ib | fosA-like | blaIMP-48, blaOXA-10 | |
| R10266 | 16 | aph(3′)-IIb-like | fosA | ||
| R10267 | 64 | aph(3′)-IIb-like | fosA | ||
| R10268 | 4 | aph(3′)-IIb, aadA6, aadA1, ant(2″)-Ia, aac(6′)-Il, aac(6′)-Ib4 | fosA | blaGES-1, blaOXA-2 | |
| R10269 | 1 | aph(6)-Ia, aph(6)-Id, aph(3′)-IIb-like, aadA11, ant(2″)-Ia | fosA-like | ||
| R10272 | 8 | aph(3′)-IIb-like, aadA6, aadA2, aadA1, ant(2″)-Ia, aacA16 | fosA-like | blaVEB-1, blaOXA-10 | |
| R10274 | 1 | aph(3′)-IIb | fosA | ||
| R10275 | 2 | aph(3′)-IIb-like | fosA-like | ||
| R10378 | 2 | aph(3′)-IIb-like | fosA | ||
| R9333 | 1 | aph(3′)-IIb | fosA | ||
| R10271 | 1 | aadA16-like, aac(6′)-Ib4 | blaKPC-2 | ||
| R10273 | 4 | aph(3′)-IIb-like, ant(2″)-Ia | fosA | ||
| R8381 | 2 | aph(3′)-IIb-like | fosA | ||
| R9010 | 8 | aph(3′)-IIb-like | fosA-like | ||
| R9042 | 2 | fosA | |||
| R10155 | 128 | aph(3′)-Iib, ant(2″)-Ia, aac(6′)-Ib | fosA-like | blaIMP-48, blaOXA-10 | |
| R10270 | 2 | aph(6)-Ia, aph(6)-Id, aph(3′)-Iib | fosA | ||
| 9260#1 | 8 | aph(3′)-IIb-like, aac(3)-IIIa | fosA | ||
| 9275#1 | 64 | aph(3′)-IIb-like, aadA2, aac(6′)-Ib | fosA-like | blaCARB-2 |
TABLE 3.
β-Lactam resistance mechanisms among the Pseudomonas aeruginosa isolates, including the isolates used for time-kill assays
| Isolate number | MLSTa (allele designation) | Mutation-driven resistance mechanisms (difference of expression compared to susceptible baseline) | Acquired β-lactamase genes | MIC (μg/ml) |
|||||
|---|---|---|---|---|---|---|---|---|---|
| CZA | MEM | AMK | COL | AZT | FOS | ||||
| 9647 | 245 (39, 6, 12, 11, 3, 15, 2) | AmpC overexpression (300×) | 1 | 1 | 8 | 1 | 16 | 128 | |
| 10270 | 357 (2, 4, 5, 3, 1, 6, 11) | Amp× overexpression (440×), OprD loss | 2 | 8 | 8 | 2 | 64 | >512 | |
| 9010 | 919 (15, 5, 1, 11, 4, 4, 10) | MexAB-OprM overexpression (6.5×) | 8 | 16 | 32 | 2 | 16 | >512 | |
| 9316 | 389 (17, 22, 5, 3, 1, 14, 3) | MexAB-OprM overexpression (6.8×) | 8 | 32 | 1 | 2 | 64 | 64 | |
| 10272 | 235 (38, 11, 3, 13, 1, 2, 4) | OprD loss | blaVEB-1, blaOXA-10 | 8 | 8 | >64 | 1 | 128 | 128 |
| 10266 | New (35, 5, 12, 11, 4, 15, 10) | MexAB-OprM overexpression (7×), OprD decrease | 16 | 32 | >64 | 1 | 64 | 64 | |
| 10267 | New (16, 10, 5, 3, ∼64, 42, 7) | MexAB-OprM overexpression (9.7×), AmpC overexpression (1,060×), OprD loss | 64 | >64 | 4 | 1 | >256 | 128 | |
| 10149 | New (111, 30, 64, 26, ∼30, 59, 7) | MexCD-OprN overexpression (5×), OprD loss | blaIMP-48, blaOXA-10 | 128 | >64 | 4 | 2 | 64 | >512 |
| 10155 | New (111, 30, 64, 26, ∼30, 59, 7) | OprD loss | blaIMP-48, blaOXA-10 | 128 | >64 | >64 | 4 | 64 | >512 |
MLST, multilocus sequence type.
The fold reductions in baseline CZA MICs as a result of the combination with the ancillary antibiotics are listed in Fig. 1. CZA MIC values against the 21 K. pneumoniae strains and the 21 P. aeruginosa strains ranged from 0.5 to 4 μg/ml and 1 to 128 μg/ml, respectively. The results of introducing the adjunctive antimicrobial on the CZA MIC values are shown in Table 1. All combinations had an MIC of ≤0.13 μg/ml in at least one strain of K. pneumoniae, whereas the CZA tested alone had the lowest MIC value, 0.5 μg/ml. The mean fold reduction was greatest when CZA was combined with MEM (12.2 ± 14.5) for the K. pneumoniae strains. This combination revealed about a 4-fold decrease in MIC in 16 strains (76.2%) out of 21. In descending order, MEM, aztreonam (AZT), COL, and FOS decreased the MIC value of CZA as well in the K. pneumoniae group. The combination with MEM was shown to have the highest fold reduction in the P. aeruginosa isolates. When combined with amikacin (AMK), CZA showed an MIC at least 2-fold lower in 17 (81%) of the P. aeruginosa group isolates. Overall, there was a greater reduction in the CZA MIC when an adjunctive antimicrobial was added in the K. pneumoniae group compared to the P. aeruginosa group. Two K. pneumoniae strains (R9009 and R10506) and two P. aeruginosa (R9647 and R10270) strains were randomly selected for time-kill analysis; results are depicted in Fig. 2a and b and Fig. 2c and d, respectively.
FIG 1.
MIC reductions of CZA in combination with adjunctive antimicrobials against K. pneumoniae and P. aeruginosa. CZA, ceftazidime-avibactam; AMK, amikacin; AZT, aztreonam; COL, colistin; MEM, meropenem; FOS, fosfomycin. Numbers above each bar represent the amount of MIC50 for that combination. The MIC ranges (in μg/ml) for K. pneumoniae are as follows: CZA-AMK, 1 to 32; CZA-AZT, 1 to 16; CZA-COL, 1 to 32; CZA-MEM, 1 to 64; and CZA-FOS, 1 to 16. The MIC ranges (in μg/ml) for P. aeruginosa are as follows: CZA-AMK, 1 to 8; CZA-AZT, 1 to 8; CZA-COL, 1 to 16; CZA-MEM, 1 to 64; and CZA-FOS, 1 to 8.
FIG 2.
Change in log10 CFU/ml from the most active single antimicrobial against K. pneumoniae R9009 and R10506 (a and b) and P. aeruginosa R9647 and R10270 (c and d). Twenty-four-hour time-kill curve graphs show only the most active concentrations of the adjunctive antibiotics. Concentrations of each antibiotic are listed in the tables (i.e., 1/2 MEM = half MIC of MEM).
Time-kill analysis.
The 24-h time-kill results are displayed in Fig. 2. The time-kill studies demonstrated that there was synergy in both of the K. pneumoniae strains when CZA was combined with AMK, AZT, and FOS, with a decrease of ≥2.82 log10 CFU/ml, while MEM and COL in combination with CZA showed synergy in only one of the two K. pneumoniae strains studied. AMK was synergistic only when the concentration equaled the MIC and was not significantly effective at 0.5× the MIC.
Time-kill analysis also revealed that both P. aeruginosa strains showed a decrease of ≥2.15 log10 CFU/ml when the combination included AMK, AZT, or MEM. These antibiotics were synergistic only when the concentration was equal to the MIC against the R9647 strain. AZT was effective when the concentration equaled 0.5× the MIC against strain R10270. The combination of AZT and CZA was also the most active combination against both Pseudomonas strains. When CZA was combined with COL, a synergistic effect was shown for R10270 (decrease of 2.15 log10 CFU/ml). Combination with FOS and CZA was indifferent in both the Pseudomonas strains tested. Combination of CZA with AMK or AZT decreased the log10 CFU/ml, demonstrating synergy against all four strains tested.
DISCUSSION
In this study, we investigated 5 novel antimicrobial combinations with CZA to assess potential synergy against 21 Klebsiella pneumoniae strains and 21 Pseudomonas aeruginosa strains that were well characterized on a molecular basis, demonstrating a wide variety of susceptibility patterns. MIC evaluation of CZA in combination with AMK, MEM, and AZT demonstrated a great reduction of the MICs of many organisms studied, especially in the Klebsiella pneumoniae group, exhibiting possible synergy. Among the K. pneumoniae isolates analyzed, 20 out of 21 carried blaKPC, including 8 isolates carrying blaKPC-3 and 12 blaKPC-2. The remaining isolate harbored blaCTX-M-15. Additionally, 14 isolates carried aminoglycoside-modifying enzymes that inactivate AMK, and all these isolates had AMK MIC values of >2 μg/ml but MIC results at <2 μg/ml when AMK was combined with CZA, and these values were lower than the MIC values for this combination tested alone. Similar synergy was noted with MEM.
Against P. aeruginosa isolates, synergy among the tested agents was less evident, but among isolates carrying blaIMP-48, blaVEB-1, and a few others, the MICs were elevated for CZA alone or tested in combination with MEM or AZT since avibactam does not inhibit metallo-β-lactamases or VEB enzymes (25). Other isolates showing a limited synergy effect overexpressed MexAB-OprM, which is able to extrude ceftazidime, among other antimicrobial agents (26).
Time-kill analysis was performed on two strains of K. pneumoniae and two strains of P. aeruginosa. Analyzing the time-kill curves, CZA in combination with AZT or AMK showed synergy against all four organisms. In addition, MEM combination demonstrated synergy against both Pseudomonas organisms and in one Klebsiella strain. CZA with COL displayed activity in one Pseudomonas and one Klebsiella organism. The FOS combination demonstrated activity against both Klebsiella strains. The concentration at which these adjunctive antibiotics demonstrated synergy varied.
During a few of the time-kill studies, adjunctive antibiotics were synergistic at 0.5× the MIC but not at 1× the MIC. This was due to the fact that the single agent itself was bactericidal at 1× the MIC, making the study of the effect of combination not possible. The observation of synergy demonstrated between CZA with MEM or AZT was of interest. Studies have shown that the addition of AVI can restore the activity of MEM or AZT against P. aeruginosa and carbapenemase-producing Enterobacteriaceae (27, 28). Further experiments are needed in order to clarify the observed improvement in activity against these strains.
Overall, based on MIC fold reductions for CZA when used in combination, MEM demonstrated the highest MIC reduction for K. pneumoniae, followed by AMK and AZT. However, for P. aeruginosa, CZA plus meropenem had the highest CZA MIC reduction. Based on the selective time-kill analysis, CZA plus MEM followed by CZA plus FOS and then CZA plus AZT were the most synergistic combinations for K. pneumoniae R9009, while for K. pneumoniae R10506 the most synergistic combinations were CZA plus COL and CZA plus AZT. In kill curve analysis, synergy against P. aeruginosa R9647 was demonstrated at 1× the MIC for CZA plus AZT, AMK, and MEM, whereas for R10270, synergy at half the MIC was demonstrated for CZA and AZT and COL. These combinations should be further evaluated with additional strains to confirm these findings.
We recognize that one major limitation of this study was the fact that we did not include metallo-beta-lactamase producer organisms, and we are planning to evaluate those isolates in our future studies. In addition, the data found from this study are based on in vitro research, and further in vivo experiments are necessary to validate these results. Furthermore, the combination studies were of short duration using fractionated and therefore minimal antibiotic exposures; this condition does not mimic human exposures at therapeutic doses. Due to the short duration of the experiments, we were not able to evaluate the impact of these combinations on supressing the emergence of resistance. Time-kill analysis was limited to representative 4 out of the 42 organisms evaluated. The potential synergy of CZA with these antibiotics based on the MIC data and time-kill assays calls for further evaluation against a wider group of organisms with various susceptibility patterns.
In conclusion, the outcomes of this study reveal synergistic activity of several novel combinations of antimicrobials with CZA. The data demonstrate the potential for the use of CZA in combination with MEM, AMK, AZT, COL, and FOS against MDR P. aeruginosa and carbapenemase-producing K. pneumoniae. Further research is merited to clarify the mechanisms of enhanced activity between CZA with MEM and AZT as well as testing the application of these combinations in clinical settings.
MATERIALS AND METHODS
Bacterial strains and culture media.
A total of 42 strains were selected for this study. Twenty-one consisted of Klebsiella pneumoniae isolates with various resistance patterns, including KPC producers. Additionally, 21 strains of MDR Pseudomonas aeruginosa, including ESBL-producing strains, were evaluated. Mueller-Hinton broth (MHB; Difco, Detroit, MI) with 25 mg/liter of calcium and 12.5 mg/liter of magnesium was used for susceptibility testing and time-kill experiments. Tryptic soy agar (TSA; Difco) was used for bacterial colony enumeration. K. pneumoniae ATCC 27853 was used for routine quality control (QC) of CZA according to the Clinical and Laboratory Standards Institute (CLSI) (29).
Antimicrobial agents.
CZA was tested alone and in combination with the following antimicrobials: MEM, AMK, AZT, COL, and FOS. AVI was provided by its manufacturer (Allergan, Parsippany, NJ). AMK was purchased from USP, Rockville, MD. AZT was purchased commercially from APP Pharmaceuticals LLC, Schaumburg, IL. MEM was purchased commercially from AstraZeneca Pharmaceuticals LP, Wilmington, DE. Ceftazidime, COL, and FOS were purchased commercially from Sigma-Aldrich Corp., St. Louis, MO.
Susceptibility testing.
MIC values of CZA, AMK, AZT, COL, FOS, and MEM were determined in duplicate by broth microdilution at approximately 106 CFU/ml per CLSI guidelines (29). If the MIC that was obtained in duplicate was not in agreement, the MIC testing was repeated. After initial MIC testing, CZA MIC values were determined in the presence of 0.5× the MIC of each respective adjunctive antimicrobial to determine the effect of each antibiotic on the CZA MIC. To determine the MIC for single agents in combination with ceftazidime, 0.5× the MIC of the combination of the single agent plus avibactam was used. For example, for K. pneumoniae R8375, the combination of CZA-MEM was performed with CZA in the presence of 0.5× MIC of the combination of MEM-AVI (0.015 μg/ml). This was done because CZA plus 0.5× MIC of MEM (16 μg/ml) produced no growth for the entire column. MIC tests for ceftazidime alone or avibactam alone were performed to understand the contribution of each antibiotic to the combination MIC.
All samples were incubated at 35°C for 18 to 20 h prior to MIC determination (29). For every batch of MIC testing, the MIC of the control strain was also evaluated, and if the expected MIC for the control strain was not achieved, the whole batch would be repeated.
Time-kill analysis.
Synergy of CZA with MEM, AMK, AZT, COL, and FOS was determined by time-kill assays performed in duplicate in MHB as the growth medium at an initial bacterial inoculum of 106 CFU/ml. CZA was tested at 0.5× the MIC or lower if 0.5× the MIC by itself was bactericidal. The adjunctive antimicrobials were tested at 0.5× and 1× the MIC for each organism. Each agent was analyzed alone and in combination with CZA against two strains of P. aeruginosa and two strains of K. pneumoniae selected randomly from the 42-strain collection. Aliquots of 0.1 ml were obtained from each well at 0, 4, 8, and 24 h, serially diluted in 0.9% sodium chloride. Samples were plated on TSA plates with a lower limit detection of 2.0 log10 CFU/ml using automatic spiral plating (EasySpiral; InterSciences, Woburn, MA). After 18 to 24 h of growth on TSA, bacterial colonies was counted using a laser colony counter (EasySpiral; InterSciences). Time-kill curves were created by plotting mean colony counts (log10 CFU/ml) versus time to compare 24-h killing effects of monotherapy and combination antimicrobial exposure. Bactericidal activity was defined as a ≥3-log10 CFU/ml reduction from baseline. Synergy was defined as a ≥2-log10 CFU/ml increase in killing at 24 h with the combination in comparison to the most active agent alone. An antagonistic effect was reported when the combination of two antibiotics caused a lower CFU reduction than the single therapy. Indifferent was defined as no change from the most active antibiotic.
Whole-genome sequencing analysis of antimicrobial resistance genes.
Isolates were subjected to whole-genome sequencing and prepared using the Nextera XT library construction protocol and index kit (Illumina, San Diego, CA) according to the manufacturer’s instructions and sequenced on a MiSeq sequencer (Illumina) with a target coverage of 30×. FASTQ format files for each sample set were assembled independently using de novo assembler SPAdes 3.11.1 (14, 30) with K-values of 21, 33, 55, 77, and 99 and careful mode on to reduce the number of mismatches, producing a FASTA format file of contiguous sequences with the best N50 value. An in-house-designed software using the target assembled sequences as queries (31, 32) to align against over 4,000 antimicrobial resistance genes from the National Center for Biotechnology Information Bacterial Antimicrobial Resistance Reference Gene Database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA313047) was used to search for potential matches applying the criteria of >94% identity and 40% minimum coverage length. Sequences displaying 100.0% homology with the reference sequences were named according to the reference. Genes with homology of <100.0% were curated and named with suffix “-like” after the gene showing the closest homology.
Mutation-driven resistance.
Selected P. aeruginosa isolates were evaluated using quantitative real-time PCR to determine the expression of chromosomal AmpC gene (PDC), mexA, mexC, mexE, and mexX, as described previously (23). Transcription levels were considered significantly different when a 10-fold increase (AmpC only) or a 5-fold increase was noted for the test gene when compared to the baseline isolate P. aeruginosa PAO1.
OprD analysis was performed using SDS-PAGE, followed by Western blot analysis with an anti-OprD antibody as described elsewhere (30). Results were compared to PAO1 and assigned as a decrease when a band fainter than the control was observed and a loss when the OprD-corresponding band was absent.
ACKNOWLEDGMENTS
S.M., N.B.S., S.A.R., and K.C.S. have nothing to declare. M.J.R. has received grant support and has consulted or spoken on behalf of Allergan, Achaogen, Bayer, Merck, Nabriva, Paratek, Spero, and Tetraphase. M.C. is employed by JMI laboratories. JMI Laboratories contracted to perform services in 2018 for Achaogen, Inc., Albany College of Pharmacy and Health Sciences, Allecra Therapeutics, Allergan, AmpliPhi Biosciences Corp., Amplyx, Antabio, the American Proficiency Institute, Arietis Corp., Arixa Pharmaceuticals, Inc., Astellas Pharma Inc., Athelas, Basilea Pharmaceutica Ltd., Bayer AG, Becton, Dickinson and Company, bioMérieux SA, Boston Pharmaceuticals, Bugworks Research Inc., CEM-102 Pharmaceuticals, Cepheid, Cidara Therapeutics, Inc., CorMedix Inc., DePuy Synthes, Destiny Pharma, Discuva Ltd., Dr. Falk Pharma GmbH, Emery Pharma, Entasis Therapeutics, Eurofarma Laboratorios SA, the U.S. Food and Drug Administration, Fox Chase Chemical Diversity Center, Inc., Gateway Pharmaceutical LLC, GenePOC Inc., Geom Therapeutics, Inc., GlaxoSmithKline plc, Harvard University, Helperby, HiMedia Laboratories, F. Hoffmann-La Roche Ltd., ICON plc, Idorsia Pharmaceuticals Ltd., Iterum Therapeutics plc, Laboratory Specialists, Inc., Melinta Therapeutics, Inc., Merck & Co., Inc., Microchem Laboratory, Micromyx, MicuRx Pharmaceuticals, Inc., Mutabilis Co., Nabriva Therapeutics plc, NAEJA-RGM, Novartis AG, Oxoid Ltd., Paratek Pharmaceuticals, Inc., Pfizer, Inc., Polyphor Ltd., Pharmaceutical Product Development, LLC, Prokaryotics Inc., Qpex Biopharma, Inc., Ra Pharmaceuticals, Inc., Roivant Sciences, Ltd., Safeguard Biosystems, Scynexis, Inc., SeLux Diagnostics, Inc., Shionogi and Co., Ltd., SinSa Labs, Spero Therapeutics, Summit Pharmaceuticals International Corp., Synlogic, T2 Biosystems, Inc., Taisho Pharmaceutical Co., Ltd., TenNor Therapeutics Ltd., Tetraphase Pharmaceuticals, The Medicines Company, Theravance Biopharma, the University of Colorado, the University of Southern California-San Diego, the University of North Texas Health Science Center, VenatoRx Pharmaceuticals, Inc., Vyome Therapeutics Inc., Wockhardt, Yukon Pharmaceuticals, Inc., Zai Lab, and Zavante Therapeutics, Inc.
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